Oligonucleotides labeled with a plurality of fluorophores

ABSTRACT

An embodiment of the invention discloses new methods for designing labeled nucleic acid probes and primers by labeling oligonucleotides with a plurality of spectrally identical or similar dyes and optionally with one or more quencher dyes. Oligonucleotides labeled in accordance with some embodiments of the invention exhibit a detectable increase in signal, for example, fluorescent signal when the labeling dyes are separated from one another. Methods for separating the dye include cleaving the labeled oligonucleotides include using enzymes that have 5′-exonuclease activity. In one embodiment nucleic acid primers of the present invention may fluoresce upon hybridization to a target sequence and incorporation into the amplification product. Nucleic acid probes and primers of the present invention have wide applications ranging from general detection of a target nucleic acid sequence to clinical diagnostics. Major advantages of the oligonucleotides including nucleic acid probes and primers of many embodiments of the present invention are their synthetic simplicity, spectral versatility and superior fluorescent signal.

PRIORITY CLAIM

This application claims the benefit of U.S. Provisional PatentApplication Ser. No. 60/523,263 filed on Nov. 19, 2003, which isincorporated herein in its entirety.

BACKGROUND

The present invention relates in general to oligonucleotides labeledwith a plurality of spectrally identical or similar dyes and optionallyone or more quencher dyes, as well as methods of creating labeledoligonucleotides and various uses of the labeled oligonucleotides asprimers or probes for highly sensitive nucleic acid detection, includingreal-time polymerase chain reaction (PCR).

Nucleic acid polymers such as DNA and RNA are essential to thetransmission of genetic information from one generation to the next andin the routine functioning of all living organisms. Accordingly thesemolecules are the object of intense study and a number of techniqueshave been developed to study of these molecules. These methods includebut are not limited to methods for identifying the presence of aspecific polynucleotide sequences in a given sample and methods designedto measure the number of specific nucleic acid molecules originallypresent in a given sample.

Practical uses for these techniques include identifying specific speciesand relationships between various species based upon similarities inoligonucleotide sequences. Other uses include diagnosing disease byidentifying specific sequences in a given sample as indicative of agiven pathology. Still other uses, too numerous to mention, includeidentifying individuals with a predisposition for developing a specificpathology as well as assessing the efficacy of proposed treatmentregimes based on the presence of specific polynucleotides in a givenpatient's genome.

One of the most widely used and powerful techniques for the study andmanipulation of oligonucleotides is the polymerase chain reaction (PCR).PCR is a primer extension reaction that provides a method for amplifyingspecific nucleic acids in vitro. This technique was first described in1987. PCR can produce million fold copies of a DNA template in a singleenzymatic reaction mixture within a matter of hours, enablingresearchers to determine the size and sequence of target DNA. This DNAamplification technique has been widely used for cloning and othermolecular biological manipulations. Further discussion of PCR isprovided in Mullis et al., Methods Enzymol. (1987); and Saiki et al,Science (1985).

In PCR the particular stretch of DNA to be amplified is referred to asthe ‘target sequence’. The target sequence is replicated by firstbinding a complimentary ‘primer’ to a single stranded portion of thetarget polynucleotide. One PCR based technique that is particularlyuseful is Quantitative PCR (qPCR). Briefly, the mechanism of qPCR isbased on the fact that PCR amplifies a target DNA in an exponentialmanner. By running a PCR reaction and measuring the total number of DNAcopies at given points during the course of the amplification reaction,one can retroactively calculate the amount of starting DNA material.

Various methods have been developed for determining the amount of PCRproduct made without having to stop the PCR run or even to sample thereaction during a given PCR run. One such method follows the course ofthe PCR run in real time by measuring the amount of product at eachcycle of DNA synthesis. This process is referred to as real timePCR(RT-PCR). Because of its great sensitivity and because measurementscan be made with the sample still in the PCR thermocylcer, variousfluorescence-based assays that monitor the formation of PCR productshave been developed. A number of instruments and methods have beendeveloped for real-time PCR(RT-PCR). A real-time PCR instrument istypically a fluorometer built upon a thermocycler. Commerciallyavailable real-time PCR instruments include Prism7700 by ABI,LightCycler by Roche, Opticon by MJ Research, iCycler IQ by BioRad, andMX4000 by Stratagene.

An oligonucleotide used to identify a given sequence of nucleic acid byhybridizing to it, but that does not serve to amplify the sequence maybe referred to as a ‘probe’. Probes also find utility in PCR reactionswhere they are used to signal polynucleotide amplification.

Given the importance of oligonucleotide and the myriad of ways in whichthese molecules can impact human, animal and plant life there is a needfor ever more efficient methods for the study and manipulation ofoligonucleotide. Including new techniques for efficiently producinglabeled oligonucleotide. One object of the present invention is toprovide labeled oligonucleotide and efficient methods for making andusing the same.

SUMMARY

The present invention provides a methods for labeling oligonucleotidesthat find utility in assays such as those designed to identify thepresence of a given polynucleotide in a sample or to amplify the amountof a given oligonucleotide or polynucleotide in a given sample. Itprovides methods for using these types of labeled oligonucleotides.

One embodiment includes oligonucleotides labeled with at least twophotometric molecules that have excitation wavelengths that are within15 nm of one another. In some embodiments the labeled oligonucleotidesproduce relatively little spectral signal until at least two of thephotometric molecules are permanently separated from each other as aresult of oligonucleotide cleavage.

One embodiment is an oligonucleotide labeled with two spectrally similaror identical photometric molecules that are fluorescent. The detectableemission from the fluorescent molecules increases when at least two ofthe molecules are permanently separated from one another as a result of-oligonucleotide cleavage. In another embodiment the florescence signalproduced by the at least two fluorescent molecules attached to theoligonucleotide increases when the oligonucleotide hybridizes to atarget oligonucleotide sequence.

One embodiment is an oligonucleotide labeled with at least twophotometric molecules in which the oligonucleotide sequence issubstantially devoid of secondary structure, such as hairpin loops andstem-loop structures.

One embodiment includes an oligonucleotide labeled with photometricmolecules that have identical or similar spectral properties and areattached at the 5′ and 3′ ends, respectively of the oligonucleotide. Inone variation of this embodiment one dye is attached at the 5′ terminalbackbone phosphate and the other dye attached at the 3′ terminalbackbone phosphate.

In one embodiment the oligonucleotide is suitable for use as primercomprising at least two spectrally identical or similar fluorescent dyesand optionally one or more fluorescence quencher dyes. In one embodimentthe oligonucleotide is suitable for use as a labeled primer. In anotherembodiment the fluorescent molecules may be attached to the bases ofnucleosides, or to a combination of the 5′ terminal backbone phosphateand the bases.

In one embodiment an oligonucleotide is labeled with at least twospectrally identical or similar fluorescent dyes. The oligonucleotidemay be a primer for use in nucleic acid amplification reactionsincluding, for example, spectrally similar or identical fluorescent dyesattached to the 5′ terminal phosphate backbone and the base of anucleoside, for example, a thymidine nucleotide.

One embodiment includes methods for producing oligonucleotides that arelabeled with at least two photometric molecules in which the photometricmolecules are spectrally similar or identical. Labeled oligonucleotidesproduced using some of these methods produce more detectable signal whenat least two of the at least two photometric molecules are permanentlyseparated from one another.

Yet another embodiment includes methods for utilizing oligonucleotideslabeled with at least two photometric molecules that have excitationwavelengths that are within 15 nm or one another's.

Still other embodiments includes uses for oligonucleotides labeledaccording to embodiments of the invention. These uses include, but arenot limited to, assays to analyze biological samples comprising nucleicacid sequence in a variety of contexts. One exemplary applicationincludes, fluorescence in situ hybridization (FISH), whereinoligonucleotide labeled in accordance with the invention can be used forlocalizing and determining the relative abundance of a target nucleicacid sequences with biological importance in, for examples, live cells,fixed tissue or a chromosome sample.

Other embodiments include using oligonucleotides labeled with aplurality of dyes in solution-based or chip-based array detectionsystems and quantification of differential expression of genes linkedwith disease in basic research and the diagnosis of disease.

Still other embodiments includes methods and kits suitable for makingoligonucleotides labeled according to embodiments of the invention.These kits may be used in the detection of amplified oligonucleotidesequences, which includes iso-thermo amplifications, ligase chainreactions and the like.

Various embodiments are suitable for use with PCR to detect biomoleculesother than nucleic acids by using an oligonucleotide-antibody conjugatewherein the antibody is specific for the biomolecules to be detected.

Further forms, embodiments, objects, functions and aspects from thepresent invention shall become apparent from the description containedherein.

BRIEF DESCRIPTION OF THE FIGURES AND TABLES

FIG. 1 is a schematic amplification plot for a typical real time PCR runthat illustrates some of the parameters associated with real time PCR.

FIG. 2A shows plots of data collected when the MCG gene was amplifiedusing PCR. These data were collected using a 6-ROX-labeled probe variousat starting concentrations of plasmid DNA. (Example 2)

FIG. 2B shows plots of data collected when the MCG gene was amplifiedusing PCR. These data were collected at various concentrations of humangenomic DNA using a 6CR110-labeled probe. (Example 3)

FIG. 2C shows plots of data collected with the MCG gene being amplifiedusing PCR. These data were collected from various startingconcentrations of human cDNA using a 6CR110-labeled probe. (Example 3)

FIG. 2D shows the amplification plots of MCG gene amplified from 100,000copies of plasmid DNA using a non-sulfonated cyanine-labeled probe.(Example 2)

FIG. 3A illustrates data collected by comparing a TaqMan probe with aprobe made in conformity with one embodiment of the invention. TheTaqMan probe was labeled with JOE at 5′ terminus and TAMRA at 3′terminus; the probe of the present invention has the sameoligonucleotide sequences as the TaqMan probe but it was labeled withR6G at both 5′ and 3′ termini. (Example 4)

FIG. 3B shows the result of a comparison of a TaqMan probe with a probemade in conformity with one embodiment of the present invention. TheTaqMan probe was labeled with FAM at 5′ terminus and TAMRA at 3′terminus; the probe of the present invention has the sameoligonucleotide sequences as the TaqMan probe but it was labeled with6-CR110 at both the 5′ and 3′ termini. (Example 4)

FIG. 3C shows the result of a comparison of a TaqMan probe with a probemade in conformity with one embodiment of the present invention. TheTaqMan probe from ABI with an undisclosed sequence had a FAM at 5′ end,a MGB and a quencher at the 3′ end; the probe of the present inventionwas labeled with 6CR110 at both the 5′ and 3′ termini. (Example 4)

FIG. 4A shows the absorption spectra of a GAPDH probe labeled with twomolecules of 5-CR110 at the termini both before and after S1 digestion(SEQ ID No. 3, Example 5).

FIG. 4B shows the absorption spectra of a GAPDH probe labeled with one5-CR110 at the 5′ terminus (SEQ ID No. 10) both before and after S1digestion. (Example 5)

FIG. 4C shows the absorption spectra of a GAPDH probe labeled with one5-CR110 at the 3′ terminus (SEQ ID No. 11) both before and after S1digestion. (Example 5)

FIG. 4D shows the amplification plots collected using the three probesused in 4A, 4B and 4C. (Example 6)

FIG. 5A shows the absorption spectra of linear and stem-looped GAPDHprobes labeled with two 6-CR110. This figure illustrates that stem-loopstructures facilitate 6-CR110-dimer formation. (Example 7)

FIG. 5B shows the absorption spectra of linear and stem-looped GAPDHprobes labeled with two molecules of 6-TAMRA. These data illustrate thata stem-loop structure strongly promotes 6-TAMRA-dimer formation.(Example 7)

FIG. 5C shows absorption spectra of a linear probe and stem-loop probefor GAPDH probes labeled with two molecules of 6-ROX. The data depictedin this figure illustrates that a stem-loop structure strongly promotes6-ROX-dimer formation. (Example 7)

FIG. 6A depicts the result of comparing the performance of a doubly6-CR110-labeled linear probe and a stem-loop probe in real time PCR atvarious probe concentrations. This figure illustrates that the linearprobe generates a significantly stronger signal than the probe thatforms a stem-loop structure. (Example 8)

FIG. 6B illustrates a comparison of the performance of a doubly6-TAMRA-labeled linear probe and a stem-loop probe in real time PCRmeasured at various probe concentrations. This figure illustrates thatthe linear probe generates a significantly stronger signal than theprobe that forms a stem-loop structure. (Example 8)

FIG. 6C depicts a comparison of the performance of doubly a6-ROX-labeled linear and a stem-loop probe in real time PCR with variousprobe concentrations. This figure illustrates that the linear probegenerates a significantly stronger signal than the probe that forms astem-loop structure. (Example 8)

FIG. 7 illustrates the chemical equilibria involved in nucleic aciddetection using a beacon probe. (Example 8)

FIG. 8A depicts the spectra of three probes labeled with a combinationof FAM/FAM, FAM/CR110 and CR110/CR110 dye pairs, respectively (Example9).

FIG. 8B illustrates amplification plots of the GAPDH gene detected withthe probes depicted in FIG. 8 k (Example 9)

FIG. 9A illustrates data collected from amplifying the GAPDH gene by PCRand detected the product with doubly 5-CR110-labeled forward primer ofGAPDH (SEQ ID No. 15). (Example 10)

FIG. 9B illustrates data collected from amplifying the GAPDH gene by PCRand detected the product with doubly 5-CR110-labeled forward primer ofGAPDH (SEQ ID No. 16). (Example 10)

FIG. 9C illustrates data collected from amplifying the GAPDH gene by PCRand detected the product with 5-CR110-labeled reverse primer (SEQ ID No.17). (Example 10)

FIG. 9D illustrates data collected from amplifying the GAPDH gene by PCRand detected the product with doubly 5-CR110-labeled forward primer (SEQID No. 15) and the same forward primer singly labeled with 5-CR110 at 5′end (SEQ ID No. 18). This graph demonstrates that only the doublylabeled oligonucleotide functions as a fluorogenic primer. (Example 10)

FIG. 9E is a comparison of data collected by amplifying the GAPDH geneby PCR and detecting the product with a doubly 5-CR110-labeled forwardprimer (SEQ ID No 16) and the same forward primer singly labeled at 3′end (SEQ No 19). This graph demonstrates that only a doubly labeledprimer functions as a fluorogenic primer for PCR detection. (Example 10)

FIG. 9F illustrates a possible secondary structure that may allow a 5′ Gto act as a quencher as in a LUX primer. This figure also illustratesthat fluorogenic primers of some embodiments of the present inventionmay work in accordance with a mechanism different from that which isresponsible for the functioning of the LUX primers. (Example 10)

FIG. 10 illustrates SNP typing of model estrogen receptors. (Example 11)

FIG. 11 illustrates data that was collected by amplifying the HCV geneby PCR and detecting the products using a doubly ROX-labeled probe (SEQID No. 33). As illustrated in the figure the signal was enhanced bytreating the product with an enzyme, which exhibits an exo-minusactivity, such as Taq DNA polymerase. (Example 12)

TABLE 1. A concise listing of some of the sequences referred tothroughout the text as well as the structures of some of linkingmolecules suitable for practicing some embodiments.

TABLE 2. A partial listing of reactive groups including someelectrophilic and nucleophilic groups that can be used in someembodiments to attach labeling molecules and quenching molecules tooligonucleotides.

SEQUENCE LISTING. An attached set of pages listing some of the sequencesused in greater detail.

DETAILED DESCRIPTION

For the purposes of promoting an understanding of the principles of theinvention, reference will now be made to the embodiments describedherein and specific language will be used to describe the same. It will,nevertheless, be understood that no limitation of the scope of theinvention is thereby intended. Any alterations and further modificationsin the described devices, systems, and treatment methods, and anyfurther applications of the principles of the invention as describedherein, are contemplated as would normally occur to one skilled in theart to which the invention relates. While aspects of the invention maybe discussed in terms of specific or general theories or principles, theinvention is in no way bound by these theories or principles. Suchdiscussion is purely illustrative and in no way limiting.

Because nucleic acid polymers play an essential role in modern medicineand the life sciences, a wide variety of reagents, including fluorescentdyes have been developed for use in processes for detecting, sequencingand measuring nucleic acid polymers. Similarly, a wide variety ofmethods have been developed for using these dyes to create ever moresensitive nucleic acid assays. One area that has generated intense studyis the development of extremely sensitive assays for measuring theaccumulation of nucleic acid polymers produced by PCR.

DNA amplification via PCR may be quite complex as the process isaffected by many factors. One factor that effects PCR is the gradualdepletion of starting components such as dNTP, primers, the effectiveconcentration of Mg²⁺ that occurs as the reaction progresses. Anotherfactor is the inhibitory effect of the accumulating end product. Duringthe PCR process these various components interact with each other in aninterrelated dynamic fashion. Typically DNA amplification comprisesthree sequential phases: the exponential phase, the linear phase and theplateau phase. Each phase is different and may exhibit a differentamplification efficiency than the other two. As a practical matter onlydata collected during the exponential phase can be used to reliablyestimate the initial concentration of target oligonucleotide.

One method for monitoring PCR in real time that often uses fluorescentmolecules is known commercially as the TaqMan assay. TaqMan assaysexploit the 5′-exonuclease activity of the Taq polymerase to monitoringDNA amplification in real time. Further discussion of this well knownassay is provided by Holland et al., Proc. Natl. Acad. Sci. USA (1991);Lee et al., Nucleic Acids Res. (1993); and U.S. Pat. Nos. 5,210,015;5,538,848; 6,258,569 and 5,691,146). The TaqMan assay detects theaccumulated PCR product via hybridization and subsequent cleavage of afluorogenic probe (the “TagMan” probe) during the amplificationreaction. The probe is an oligonucleotide whose sequence iscomplementary to the target DNA to be detected. The probe is labeledwith a single fluorescent reporter dye and a single fluorescencequencher. The reporter and quencher dyes are attached to theoligonucleotide with a separation of typically about 15 to about 60nucleotides for optimal fluorescence quenching and 5′-exonucleaseactivity. The dye can be attached to either the nucleotide bases or thebackbone of the oligonucleotide or a combination thereof. Typically, oneof the reporter/donor labels is attached to the 3′ terminal backbonephosphate and the other attached to the 5′ terminal backbone phosphate.The quencher can be either a non-fluorescent dye or a fluorescent dye ofappropriate wavelengths. In both cases, quenching of the fluorophorebound to the probe occurs via fluorescence resonance energy transfer(FRET).

In order to briefly discuss how to select a fluorophore/quencher pairand design a TaqMan probe that will exhibit optimal performance, it isuseful to review some basic photophysics. Firstly, when a dye receives aphoton of sufficient energy from an external source, it iselectronically excited to the singlet excited state. The excited stateexists for a finite time, during which period the electronic energy ispartially dissipated into vibrational and rotational modes of themolecule. Consequently, when the dye emits the photon, the energy of thephoton is lower and thus the wavelength of the photon emitted by themolecule is longer than the wavelength of the photon absorbed by themolecule. This energy or wavelength difference between excitation, orabsorption, and emission is called the Stokes shift and is common amongfluorescent dyes. Secondly, FRET-based quenching is governed byFörster's resonance energy transfer theory, which states that theefficiency of photo energy transfer is positively related to the overlapof emission spectrum of the donor and absorption spectrum of theacceptor. Further discussions of this is provided in Förster, Ann. Phys.(1948); and Stryer et al., Proc. Natl. Acad. Sci. (1967). Thecombination of the Stokes shift and the spectral overlap requirementnecessitates that the fluorescent donor and acceptor be different dyes.This is true even if the donor and quencher molecules are bothfluorescent dyes.

In fact, a quencher molecule that is also a fluorescent dye shouldideally be selected to have a wavelength of emission that issignificantly longer than the emission wavelength of the donor(reporter) dye. Using this combination ensures a dark background. Anadditional gain in sensitivity can be achieved by using a filter thatselectively blocks all or most of the signal emitted by the quenchermolecule. For optimal performance then, quenchers are preferably chosenfrom molecules that are themselves completely non-fluorescent. Indeed,achieving a low or zero background for TaqMan assays has been the focusof considerable research effort. To this end, many non-fluorescentquencher molecules have been developed. Well known commerciallyavailable non-fluorescent quenchers include, for example, Black HoleQuencher (BHQ) dyes from Biosearch, which are non-fluorescent azo dyesand Eclipse Dark Quencher (DQ) from Epoch (Eurogentec catalog numberOL-0273-DQ02), and IOWA Black (IWB) from Integrated DNA Technologies.Further discussion of these molecules is provided by Johansson, M. K, etal, J. Am. Chem., Soc., (2002). These and similar non-fluorescentquenchers improve the sensitivity of TaqMan probes by suppressingbackground fluorescence, thereby increasing the signal gain followingenzymatic cleavage of the probe. Examples of popular donor/acceptor(fluorophore/quencher pairs used in TaqMan probe design includeFAM/TAMRA, VIC/BHQ1, HEX/BHQ2, and TET/DHQ and the like.

In addition to the spectral overlap requirement stated above, a secondparameter critical to FRET-based fluorescence quenching is the distancebetween the donor and acceptor. FRET is dependent on the inverse sixthpower of the intermolecular separation.

Prior to hybridization with a target nucleic acid, a typical TaqManprobe assumes a random conformation, wherein the donor and quencher areable to move close enough to one another for efficient FRET to occur.Because of their proximity to one another and their spectralcharacteristic the reporter molecule, is quenched and therefor theprobe, is non-fluorescent. Upon hybridization with the segment of atarget DNA being amplified, the probe is stretched and consequentlybecomes fluorescent because the increased distance between the donor andquencher makes FRET energy transfer impossible. Alternatively, and oftenpreferably, the probe is digested during amplification by, for example,using a polymerase that has 51-exonucleas activity. Digestion completelyfrees the donor dye from being in proximity to the quencher, therebyfurther reducing quenching and further enhancing the fluorescence signalfrom the donor dye. As more DNA copies are made during the PCRamplification, more probes are hybridized and then cleaved, which inturn increases the fluorescence signal A variation of this scheme is theuse of a two-enzyme system. In this system one enzyme is responsible forpolymerizing the oligonucleotide and the second enzyme is responsiblefor cleaving the reporter dye from the PCR product. One suchcommercially available product that exploits this strategy is the FullVelocity kit available from Stratagene.

Another variation of probe design that exploits FRET to create probeswith good signal to noise ratios is described in U.S. Pat. No.6,492,346. This variation employs a minor groove binding molecule (MGB)covalently linked to the 3′-end, 5′-end, or any other position withinthe oligonucleotide probe. Probes containing a MGB may have a shorternucleotide sequence than probes that do not use MGBs and still exhibitvery efficient fluorescence quenching. In addition, probes labeled witha MGB tend to have improved specificity, efficiency and enhanceddiscrimination against mismatches relative to probes labeled by certainother labeling molecules that do not selectively bind to the minorgroove.

Still another method of detecting amplification products uses theso-called “molecular beacon probe” which is the subject of U.S. Pat.Nos. 5,925,517; and 5,118,801 and 5,312,728. Molecular beacon probeshave stem-loop structures including a central target-recognitionsequence (loop region) that is flanked by a pair of complimentary 3′ and5′ terminal sequences that hybridize to each other to form a stemstructure. A fluorescence donor dye and quencher molecule are attachedto the 3′ and 5′ terminals, respectively. In the absence of acomplementary target sequence the beacon probe stays in its closedconformation, and the fluorescence of the donor dye is quenched by thequencher dye via FRET. Upon hybridization with a target sequence, thebeacon probe stretches to an open conformation, thereby separating thedonor dye and quencher, and increasing the fluorescence signal of thedonor dye. As additional copies of a target DNA are generated during thecourse of a PCR reaction, more beacon probes adopt the open conformationby hybridizing to the target DNA and the fluorescence signal risesaccordingly. Further discussion of this technique is provided by Tyagiet al., Nature Biotechnol. (1996). Unlike TaqMan probes, which are“consumed” via enzymatic cleavage of the probes during PCR, molecularbeacon probes remain chemically unchanged throughout the DNAamplification process; only the conformation of the beacon probeschanges from the closed form (stem-loop) to the open form.

The two conformations of the beacon probes—open and closed—exist in adynamic equilibrium with one another. During PCR the equilibrium dependson three competing hybridization reactions between each pair ofcomplementary strands. The three equilibria are between: 1) the twocomplementary strands of the target DNA; 2) the complementary 3′ and 5′ends (stem) of the probe; and 3) the target-recognition sequence of theprobe (loop) and the target sequence. Only the third hybridizationreaction favors the open beacon conformation and thus only one producesa detectable fluorescence signal. Both the first and secondhybridization reactions favor the closed beacon conformation and in turnreduce the fluorescence signal. Because of the competing hybridizationinteractions (1 and 2), a molecular beacon probe is inherently lesssensitive in nucleic acid detection than a TaqMan probe labeled evenwhen both are labeled with the same or similar donor/quencher dye pair.However, a beacon probe hybridized to a target sequence can also becleaved from the oligo as is a TaqMan, probe if an enzyme with5′-exonuclease activity is used in the reaction. In this instance, abeacon probe has become a de facto TaqMan probe.

Still another method is disclosed in U.S. Pat. No. 6,174,670, thismethod uses an energy transfer system in which energy transfer occursbetween two hybridization probes. For example, the first probe islabeled at the 3′ end with a fluorescence donor dye, while the secondprobe is labeled at the 5′ end with a acceptor dye. The acceptormolecule on the second primer emits energy at a longer wavelength thanthe donor dye on the first primer. When employed in PCR, the two probeshybridize to one of the two complementary strands of a target DNA in ahead to toe arrangement. Because of how the two donor and acceptor dyesare positioned FRET occurs between these two molecules. Accordingly, bymeasuring the fluorescence emission of the acceptor dye, one can relatethe fluorescence intensity to the amount of a target DNA being generatedas the PCR progresses.

Yet another method of detecting amplification products is disclosed inU.S. Pat. No. 6,635,427. This method uses an internal guanosine (G)nucleotide as a quencher (acceptor) for a single reporter dye attachedto an oligonucleotide probe. In the absence of a target sequence, theguanosine nucleotide, which is usually strategically positioned near tothe position of the reporter dye, quenches the fluorescence of thereporter dye. Upon hybridization with the target sequence, quenching byguanosine is reduced, leading to an increase in the fluorescence signal.

PCR can also be monitored by using of fluorogenic primers that becomefluorescent upon incorporation into the amplification products. One suchmethod that uses a beacon-like hairpin primer labeled with adonor/quencher dye pair is known commercially as Ampliphore, and isdisclosed in U.S. Pat. No. 5,866,336. Prior to hybridizing to its targetDNA sequence, the primer exists in the closed hairpin conformation,which quenches the fluorescence of the donor dye via FRET. Oncehybridized to the target DNA sequence and incorporated into theamplification product, the primer assumes an extended open conformationand the primer becomes fluorescent because FRET is diminished. In orderto achieve the required specificity of a primer as well as to maintainthe necessary hairpin structure, a beacon-like primer is usually quitelong. The length of the probe imposes restrictions on primer design andadds to the cost of synthesizing the primer.

Another method of monitoring PCR uses a primer known commercially as theLUX primer. Further discussion of this technique is provided byNazarenko et al., published in Nucleic Acid Research (2002); and -Marraset al., Nucleic Acid Research (2002). LUX primers are labeled with asingle fluorescent dye (donor molecules) positioned strategically nearan internal guanosine (G) nucleotide that acts to quench thefluorescence of the dye. The labeled primer in its free form isnon-fluorescent or weakly fluorescent due to the interaction between thedye and the G nucleotide that is near to it. When the primer is extendedand the G nucleotide is internalized in double stranded DNA,fluorescence quenching is reduced or eliminated and the fluorescentsignal from the donor molecule increases. LUX primers have the advantageof being relatively simple in design. One problem with the LUX system isthat it requires the presence of a G nucleotide near the dye attachmentsite (usually thymidine) this restricts choices in primer design.Furthermore, the fluorescence quenching by G only works well with a veryfew dyes of selected wavelength. The restriction on primer sequence and,especially, the incompatibility with longer wavelength dyes make itdifficult to develop a set of LUX primers for use in multiplex PCR.

More recently, BD Biosciences developed a fluorescence-based primer kitknown commercially as DNAzyme for use in RT-PCR. This method employs acombination of a fluorogenic oligonucleotide and primers and isdisclosed in US Patent Application Publication No. 2001/0001063. Inaddition to the normal initiation sequence for the extension reaction,one of the primer sequences encodes an enzyme that cleaves thefluorogenic oligonucleotide during primer extension. The result, as withthe TaqMan assay, is in an increase in the fluorescence signal. Thefluorogenic oligonucleotide used in the primer kit is similar in designto a typical TaqMan® probe except that the former does not have asequence complimentary to the target sequence and as a result thistechnique lacks the specificity of TaqMan based assays. Furthermore,because of the need to code for the requisite enzyme, the DNAzyme primercan be easily over 50 nucleotides long. The need for a longer nucleotideincreases the cost of manufacturing the primer.

One major advantage of fluorogenic probes over fluorogenic primers isthat fluorescence signal detected from probes derives only fromhybridization between probe and target. Non-specific amplification ofsignal due to mis-priming or primer-dimer artifacts, as sometimes occurswith primers and does not generate useful signals, does not generallyoccur with probes. Fluorogenic probes can be labeled with different,distinguishable reporter dyes. By using several probes each labeled witha unique reporter dye, amplifications of multiple targets with distinctsequences can be detected in a single PCR reaction. This method iscommonly referred to as multiplex PCR. The development of fluorogenicprobes has also made it possible to eliminate post-PCR processing,thereby eliminating the possibility of cross-contamination, which is acritical factor for clinical diagnostics and forensic applications.

One disadvantage of fluorogenic probes is their cost. A fluorogenicoligonucleotide is at least 10 times more expensive than thecorresponding unlabeled oligonucleotide. Furthermore, probe design isrelatively complex and one often needs to consider many factors such asthe length of probe, annealing temperature and proper spectral matchingof quencher to fluorophore when constructing a suitable probe.

Compared with fluorogenic oligonucleotide probes, particularly TaqManprobes, fluorogenic primers tend to generate a weaker signal becausethey are not cleaved during PCR to allow permanent separation of thereporter dye from the quencher. Additionally, non-specific PCR productsresulting from mis-priming and primer dimer formation also contribute toincreasing noise in the assay. For these reasons, fluorogenic probes aregenerally preferred over fluorogenic primers for use in real timequantitative PCR.

Numerous methods have been developed for labeling oligonucleotides.Typically a fluorescent donor dye and a quencher are attached to theoligonucleotides in a stepwise fashion. These processes often involveexpensive reagents and complex protection/de-protection steps and theyields are often quite low. Moreover, the choice of dyes and quenchersas well as the order in which they are attached to the oligonucleotidesare limited and inflexible, in part, because not all dyes can toleratethe harsh chemical conditions of oligonucleotide syntheses.

Typically, a dye is incorporated into an oligonucleotide via one of twomethods: 1) by using a dye-modified nucleoside or deoxynucleosidephosphoramidite during automated synthesis; or 2) in a post-synthesislabeling by reacting an amine- or thiol-modified nucleotide ordeoxynucleotide with an amine- or thiol-reactive dye. For example, anoligonucleotide labeled with a FAM/TAMRA dye pair at the 3′ and 5′termini is typically synthesized by starting with a TAMRA-labeledmodifier attached to CPG solid support, followed by successive buildupof the remaining oligonucleotide. The donor dye FAM is attached to theoligo at the last coupling step via standard phosphoramidite chemistry.This approach has two drawbacks: first, dye-labeled phosphoramidite iscostly; second, it diminishes the quenching ability of TAMRA becauserhodamine dyes in general, and TARMA, in particular are unstable underthe standard oligonucleotide synthesis condition. To address thisproblem, it is a common practice to avoid starting with TAMRA linked toCPG support. One approach is to start the synthesis with a protected3′-amino-modifer linked CPG and the donor dye FAM is attached to the5′-end as usual at the last step of the automated synthesis via standardphosphoramidite chemistry. Following subsequent oligonucleotide cleavagefrom the solid support and a 3′-amine de-protection step, theamine-modified oligonucleotide is reacted with a TAMRA succinimidylester. To ensure the performance of the probe or primer, purificationsteps using either HPLC or polyacrylamide gel electrophoresis arenecessary both before and after to the second dye attachment reaction.The limitations and inflexibility imposed by the designs of the probesand primers as well as the nature of the labeling chemistry make thesefluorogenic oligonucleotides very expensive to manufacture.

An increasing important type of PCR assay that makes use of labeledoligonucleotides is real-time PCR. One important parameter for areal-time PCR monitoring is the so-called threshold cycle point, or Ctvalue. The Ct value is the theoretical number of reaction cycles neededfor the fluorescent signal of the PCR product to reach a pre-set valueabove the baseline. The higher the concentration of a target DNA in thesample, the smaller the Ct value will be. FIG. 1 shows a representativeamplification plot and defines the terms used in the quantificationanalysis. An amplification plot is the plot of fluorescence signalversus cycle number. In the initial cycles of PCR, there is littlechange in fluorescence signal. This relatively “flat region” defines thebaseline for the amplification plot. An increase in fluorescence abovethe baseline indicates the detection of accumulated PCR product. Thelogarithm of initial target copy number is reversibly correlated to Ctvalue in a linear fashion, and this relationship forms the mathematicalbasis for real-time quantitative PCR.

DEFINITIONS OF SOME OF THE TERMS USED HEREIN

The terms “oligonucleotide” and “oligo” are used interchangeably andrefer to a sequence of nucleic acids, 2′-deoxynucleic acids, peptidenucleic acids (PNA), locked nucleic acid (LNA) and other unnaturalnucleic acids which include pyrazolo pyrimidine. In generaloligonucleotides are of a length suitable for use as primers or probes.Most oligonucleotides are polynucleotides generally less than 100nucleotides long, many are less than 50 nucleotides long and a number ofoligonucleotides are comprised of 25 or fewer nucleotides. For a morethorough discussion of the term the reader is directed to the followingreferences Kutyavin, I., et al., N. A. R. 30, 2002:4952-4959; and He J.& Seela F., N. A. R. 30, 2002:5485-5496 and references therein.

“Primer” refers to an oligonucleotide that is capable of acting as astarting point to extend along a complementary strand. Primers usuallyare used as a set in PCR, one forward and one reverse. The forwardprimer contains a sequence complementary to a region of one strand oftarget nucleic acid and guides the synthesis along this strand.Similarly the reverse primer contains a sequence complementary to theopposite stand of the target nucleic and guides the synthesis along theopposite strand of target nucleic acid.

“Probe” refers to a labeled oligonucleotide containing a sequencecomplementary to a region of the target nucleic acid, wherein thelabeled oligonucleotide anneals to the target sequence and generates asignal indicating the presence of the region of the target. The probe isgenerally blocked at the 3′ terminus and is not extended into products.

The term “photometric labeling molecule” refers to molecules thatgenerate a detectable change signal due to change in the molecule'sphysical or chemical environment and used to label another molecule. Thechange may be in the amount of light absorbed or in the wavelength oflight absorbed. Photometric molecules include, for example, fluorescentdye molecules that absorb light at one length and emit light at anotherwavelength. In the case of photometric labeling molecules that arefluorescent dyes the molecules may also exhibit a change in the amountof light emitted or in the wavelength of the light emitted.

The term ‘reactive groups’ refers to chemical moieties that may beuseful in attaching various labeling groups including fluoropores andquenching molecules to oligonucleotides. The choice of reactive groupused to attach the dye to an oligo typically depends on the functionalgroup on the oligo to be labeled.

The bond formation reaction between a reactive group of, for example, adye molecule and a functional group of a oligo is typically a reactionbetween a nucleophile and an electrophile. Accordingly, a reactive groupcan be either a nucleophile or a electrophile, and correspondingly afunctional group can be either an electrophile or a nucleophile. Anon-exhaustive list of pairs of electrophile/nucleophile can be found inTable 2. For a further discussion of reactive pairs the reader isdirected to U.S. Pat. No. 6,130,101.

Typical functional groups present on an oligo include, but are notlimited to, amines, thiols, alcohols, phenols, aldehydes, ketone,hydrazines, hydroxylamines, disubstituted amines, halides, or carboxylicacids. More typical functional groups on an oligo are amines, thiols,alcohols, aldehydes or a ketones.

Common reactive groups attached to a dye molecules include, but are notlimited to: acrylamide, an activated ester of a carboxylic acid, an acylazide, an acyl nitrile, an aldehyde, an alkyl halide, an amine, ananhydride, an aniline, an arylhalyde, an azide, an aziridine, acarboxylic acid, a haloacetamide, a halotriazine, a hydrazine, ahydrazide, an imido ester, an isocyanate, an isothiocyanate, amaleimide, a phosphoramidite, a sulfonyl halide or a thiol. Otherreactive groups include succinimidyl ester, an amine, a haloacetamide, ahydrazine, an isothiocyanate, maleimide, or a phosphoramidite. When thefunctional groups present on the oligonucleotide are amines one commonlyused reactive group on the dye used to attach the dye to theoligonucleotide is a succinimidyl ester.

Some of the abbreviations used for various reagents including dyes areas follows: 5-CR110 refers to 5-carboxyrhodamine-110; 6-CR110 refers to6-carboxyrhodamine-110; 5-FAM refers to 5-carboxyfluorescein; 6-FAMrefers to 6-carboxyfiuorescein; 5-R6G refers to 5-rhodamine 6G; 5-ROXrefers to 5-carboxy-X-rhodamine; 6-ROX refers to 6-carboxy-X-rhodamine;5-TAMRA refers to 5-carboxytetramethylrhodamine; 6-TAMRA refers to6-carboxytetramethyl-rhodamine; JOE refers to2′,7′-dimethoxy-4′,5′-dichloro-6-carboxyfluorescein

The term “spectrally similar dyes”, for the purpose of the presentinvention, refers to fluorescent dyes that may or may not have similarchemical structures but possess similar excitation and/or excitationspectral properties. The emission spectra of the dyes, however, may ormay not be similar. An example of one such pair is 5-FAM and 5-CR110.5-FAM has absorption/emission wavelengths at 495/519 nm while 5-CR110has absorption/emission at 502/524 nm. For the purpose of the presentinvention, absorption or excitation wavelengths having a differencewithin 15 nm are considered to be similar.

The term “spectrally identical dyes” refers to fluorescent dyes that mayor may not have the same chemical structures but have either emissionprofiles or excitation or both emission and excitation profiles that arespectrally indistinguishable.

The term “same dyes” refers to fluorescent dyes that are both chemicallyand spectrally identical.

The term “reporter dye” or “fluorescent reporter dye” refers to afluorescent dye whose fluorescence is monitored during an assay. When aquencher dye is also used to label the same biomolecule, a reporter dyemay be referred to as a donor dye and the quencher dye may sometimes bereferred to as an acceptor, acceptor dye or acceptor molecule.

As used herein, the terms “quench” or “quenches” or “quenching” or“quenched” refer to reducing the signal produced by a molecule, itincludes, but is not limited to, reducing the signal produced to zero orto below a detectable limit. Hence, a given molecule can be “quenched”by, for example, another molecule and still produce a detectable signalalbeit the size of the signal produced by the quenched molecule will besmaller when the molecule is quenched than when the molecule is notquenched.

The term “quencher” or “quencher dye” or “quencher molecule” refers to adye or an equivalent molecule, such as nucleoside guanosine (G) or2′-deoxyguanosine (dG), which is capable of reducing the fluorescence ofa fluorescent reporter dye or donor dye. A quencher dye may be afluorescent dye or non-fluorescent dye. When the quencher is afluorescent dye, its fluorescence wavelength is typically substantiallydifferent from that of the reporter dye and the quencher fluorescence isusually not monitored during an assay.

Some embodiments of the present invention disclose methods forconstructing fluorogenic oligonucleotides and their uses as primers andprobes for the purpose of nucleic acid detection, particularly nucleicacid detection in real-time qPCR. Compared with existing technologies,fluorogenic oligonucleotides of some embodiments are significantlyeasier to manufacture and substantially more sensitive than manycommercially available technologies. Furthermore, unlike conventionalfluorogenic oligonucleotides, such as the TaqMan probes, whose choice ofa reporter dye is limited by the availability of the accompanyingquencher dye, oligonucleotides of various embodiments of the presentinvention can accommodate fluorescent dyes of virtually any wavelength.This flexibility in dye selection facilitates syntheses of fluorogenicprimers or probes of different fluorescent wavelengths and allowsmultiplex detection in a single-tube format.

Some embodiments are probes that are single-stranded oligonucleotideslabeled with a plurality of spectrally identical or similar fluorescentdyes. The probes may be further labeled with one or more quenchers. Inthe absence of a target sequence, the probes assume a random coiledconformation, and are either non-fluorescent or only weakly fluorescent.Generally, an oligonucleotide with a random coiled conformation issubstantially free of secondary structure that serves to bring the twotermini of the oligonucleotide in proximity with one another. Incontrast, an example of oligonucleotide probes having a secondarystructure is the so-called molecular beacon probe.

In the presence of a target sequence, probes of various embodimentsreadily hybridize to the target sequence. In the presence of specificreagents or enzymes with 5′ exonuclease activity the dye molecules aresubsequently cleaved from the oligonucleotide. Cleavage results inpermanent separation of the dye molecules resulting in a large increasein fluorescence.

Maximal fluorescent increase is dependent on the cleavability of thephosphate backbone linking each neighboring dye pair by the5′-exonuclease or an equivalent nuclease. When the dye attachment sitesare too close to each other, hybridization of the oligonucleotide to thetarget and 5′-exonuclease activity may be hindered. On the other hand,when the dye attachment sites are too far away from each other,background fluorescence may be high. Thus, to achieve minimalfluorescence background in the absence of a target sequence and optimal5′-exonuclease activity following probe/target hybridization, eachneighboring pair of dye attachment sites should be separated by 3 to 60nucleotides, preferably by 12 to 35 nucleotides, and most preferably by15 to 25 nucleotides.

Useful enzymes for use with these embodiments include Taq polymerase ora stand-alone exonuclease, or any other enzyme that can cut between thetwo labeling molecules. In some embodiments, permanently separating thelabeling molecules substantially increased fluorescent emission.

In contrast to the multiple labeling molecules incorporated into oligoslabeled according to the invention, fluorogenic probes such as theTaqMan probes disclosed in U.S. Pat. No. 5,538,848, Molecular Beaconsdisclosed in U.S. Pat. No. 5,925,517 and the Hyb probes disclosed inU.S. Pat. No. 6,174,670 include only single fluorescent reporter. As aresult, the maximum fluorescent signal that currently available probescan generate is the signal produced by a single fluorophore. On theother hand, probes of various embodiments of the present inventioninclude at least two dyes. Accordingly, hybridization with a targetsequence and cleavage of the probes of the present invention results inthe generation of signal from multiple fluorescent dyes. Accordingly,probes of various embodiments of the present invention are capable ofgenerating fluorescent signal many times stronger than the signalsgenerated by probes made in accordance with the currently availablemethods.

FIG. 3A illustrates the result of comparing the kinetic fluorescencemeasurements of a typical MCG gene amplification reaction detected witha TagMan probe (JOE as the fluorophore and TAMRA as the quencher) andthe same reaction detected with a probe made in accordance with oneembodiment of the present invention. The probe made in accordance withone embodiment of the instant invention was labeled with two R6G dyes atthe two termini (See Example 4). The same sequence was used in bothassays.

As the data in FIG. 3A illustrate the fluorescence signal from thedoubly labeled probe is twice that of the signal measured using theTagMan probe. It is a surprising observation that an oligonucleotidelabeled with two or more identical or spectrally similar fluorophores issufficiently quenched (without forming dye aggregates) that separationof the fluorophores from one another by cleavage of the probe generatesa signal high enough above background to be useful in tracking theamplification of DNA. Because this observation with oligonucleotides wasso unexpected, we looked for an explanation to techniques that usefluorescent dyes to measure the level of another biopolymerpolypeptides.

Proteins are sometimes labeled with antibodies tagged with fluorescentdyes. In these assays it is common to attach two or more identicalfluorophores to a single polypeptide. The additional of each dyemolecule increases the overall fluorescence of the labeled proteins,although the fluorescence increase over the number of dyes may not belinear due to fluorescence quenching related to physical touching of thedyes. In protein labeling experiments significant fluorescence quenchingoccurs, only when an excess number of dye molecules are attached to theprotein. In such cases, the quenching is often a result of the physicalinteraction among the dye molecules namely dye aggregation. Furtherdiscussion of this technique is provided in Haugland, R P Handbook ofFluorescent Probes and Research Products 9^(th) edition, pp. 20-74 andreferences therein.

In contrast to relatively highly structured polypeptides,oligonucleotides lacking complimentary internal sequences are expectedto assume unstructured, unpacked, extended structures. This conformationis generally referred to as random coil and is characterized by a lackof readily definable internal secondary structure. Oligonucleotides arebelieved to adopt the random coil conformation in order to minimizeinter molecular electrostatic repulsion due to the highly negativelycharged phosphate backbone of the molecule. Consequently, the dyes usedto label, for the example, the termini of an oligonucleotide with asubstantially random coil shape are not expected to interact with oneanother and should not be expected to demonstrate fluorescence signalquenching. Therefore, based on what is widely known about proteinlabeling and oligonucleotide structure, one would have expected thatoligonucleotides of various embodiments of the present invention wouldexhibit too little fluorescence quenching to be useful in detecting thepresence of or level of oligonucleotide in a given sample. Based on whatis taught in the art it was expected that the background fluorescence ofthese molecules would be so high that they could not be used to monitorreal-time PCR. Indeed, researchers have gone to great length to designelaborate oligo structures that position labeling fluorophores so as tofacilitate fluorophore aggregation to quench fluorescence. For example,both U.S. Pat. Nos. 6,150,097 and 6,037,137 mentioned the possibility ofdesigning a real-time PCR probe based on Molecular Beacon structure thatbrings two reporter fluorophores into physical contact with one another.

Another unexpected observation gleaned from using one embodiment of thepresent invention is that oligonucleotide secondary structure thatpromotes dye aggregation is both unnecessary and undesirable. Weobserved that aggregation of identical reporter dyes is not onlyunnecessary, but in many instances detrimental to the performance ofreal-time PCR probes. Probes of one embodiment of the present invention,that have the simplest structure, are oftentimes much more sensitivethan probes made in accordance with methods disclosed in much of theprior art. Additionally, probes that are designed to have complexinternal secondary structure are generally more difficult to design andmanufacture than are probes that are substantially devoid of internalsecondary structure.

It has been widely reported that certain fluorescent or non-fluorescentdyes tend to form ground state complexes. These complexes are likely toform, in aqueous solvents at high concentrations or when the moleculesare within close proximity to one another. Further discussion of this isprovided by West et al., J Phys Chem (1965); Rohatgi et al., J Phys Chem(1966); Rohatgi et al., Chem Phys Lett (1971); and Khairutdinov et al.,J Phys Chem. (1997).

The formation of either a homodimer between two identical dyes, orheterodimer between two different dyes leads to a distinct change in theabsorption spectrum of the dyes. This is believed to be the result ofcoupling of the excited state energies of the dyes. A particular type ofdimer called an H-dimer that forms between two fluorescent dyes orbetween a fluorescent dye and a non-fluorescent dye is characterized bya blue shift in the absorption maximum of the dyes and fluorescencequenching. Fluorescence quenching due to H-dimer formation has beenexploited to construct fluorogenic peptidase substrates. Furtherdiscussion of this subject is provided by Packard et al., Proc. Natl.Acad. Sci. (1996); Geoghegan et al., Bioconjugate Chem (2000); Tyagi etal., Nat. Biotechno. (1998); Bernacchi et al, Nucleic Acids Res. (2001);Marras et al., Nucleic Acids Res. (2002); Johansson et al., J. Am. Chem.Soc. (2002); U.S. Pat. No. 6,037,1376 and 150,097.

In all of the aforementioned references, the labeled peptides oroligonucleotides are constructed in a manner designed to ensure that themolecules are physically close enough to one another so that signal fromthe donor dye is quenched prior to enzymatic cleavage. This isapparently the case with peptidase substrates, and with molecular beaconprobes before they hybridize to their target sequences. For example, thefluorogenic peptides disclosed in U.S. Pat. No. 6,037,137, requireso-called, “conformation determining regions” that introduce bends intothe peptides so that the dye pair are held in close proximity forefficient “contact fluorescence quenching”. Similarly, U.S. Pat. Nos.6,150,097 and 6,037,137 disclose molecular beacons having a fluorescentreporter dye attached to one terminal and a quencher dye attached to theother terminal. Alternatively these probes have one reporter dyeattached to one terminal and an identical reporter dye attached to theother terminal, wherein the dye pair is in physical contact to effectfluorescence quenching. In a variation of this method one groupreportedly achieved contact quenching by labeling a linearoligonucleotide with a fluorophore and a highly hydrophobic quencherthat favors formation of a heterodimer with the fluorophore. SeeJohansson, et al, J. Am. Chem. Soc. (2002).

In contrast to these approaches, oligonucleotide probes of someembodiments of the present invention are substantially devoid of clearlydefinable secondary structure or other conformation determiningstructures that result in the probes assuming a particular rigidconformation. Furthermore, it is not necessary to form a dimer or otherstructures that facilitate physical “touching” between the dyes in theprobes of many embodiments of the present invention comprising two ormore fluorescent reporter dyes.

The absence of proximal quenching between the dye pair of variousembodiments is demonstrated by a lack of significant alteration in theabsorption profile of the doubly labeled probe before and afterenzymatic digestion. See, for example, Traces 29 vs. 28 in FIG. 4A andfurther discussion in Example 5. The small overall wavelength shift fromTrace 28 to Trace 29, is different from a change in the shape or profileof the spectrum, and is caused by a difference in the micro environmentthat the dye experiences. This is referred to as the “solvent effect”.The solvent effect is confirmed by the nearly superimposable spectra ofthe probe doubly labeled with CR110 (Trace 29) and anotheroligonucleotide of the same sequence labeled with a single CR110 ateither 5′ (Trace 31 in FIG. 4B) or at 3′ (Trace 33 in FIG. 4C). In thisinstance the dyes experience a similar solvent effect. Once again, thesimilarity in the absorption spectra between the doubly labeled probeand the two singly labeled oligonucleotides indicates that there issubstantially no dye aggregation in the doubly labeled probe.

To illustrate the spectral change upon dimer formation, we synthesizedthree molecular beacon probes with loop sequences that are identical tothat of the linear probe. One beacon probe was labeled with two 5-CR110dyes at the 3′ and 5′ terminals (SEQ ID No 12), another beacon probeswas labeled with two 5-TAMRA dyes at the 3′ and 5′ termini (SEQ ID No13. And still another beacon probe was labeled with two 5-ROX dyes atthe 3′ and 5′ termini (SEC) ID No 14). As shown in FIGS. 5A, 5B and 5Cand further detailed in Example 7. The absorption spectra of all beaconprobes (Trace 38, 40, and 42) have been altered significantly from thoseof linear probes (Traces 37, 39, and 41), with each forming a newshorter wavelength peak characteristic of H-dimer formation. For a morethorough discussion of this please see Blackman et al., Biochemistry(2002); Packard et al., Proc. Natl. Acad. Sci. (1996). Upon S1 nucleasedigestion, the spectra changed back to that of free dye. Although thefluorescence of beacon probe labeled with two reporter dyes is wellquenched as a result of dye dimer formation, the probe has relativelylow sensitivity for nucleic acid detection. FIGS. 6A, 6B, and 6Ccompares the kinetic fluorescence measurements between the molecularbeacons and the linear probe each labeled with two identical dyes of5-CR110, 5-TAMRA, 5-ROX, respectively according to various embodimentsof the present invention.

As the data indicate, the probes made according to various embodimentsof the present invention are often times 2-10 times more sensitive thanthe corresponding beacon probes. The significantly weaker sensitivity ofthe beacon probes may be explained in terms of competing equilibria thatexist during the PCR detection process. As illustrated in FIG. 7,(details in Example 8), there are three competing equilibria these arebetween: 1) single stranded target DNA and double stranded target DNA(K_(A)); 2) beacon in the open conformation and beacon in the closedconformation (K_(B)); and 3) the probe-target hybridization product andthe two reactants, single stranded target DNA and beacon in randomconformation (KO.

Formation of the probe-target hybrid separates the two dyes, and as aresult, fluorescent signal is generated. Clearly, the higher theconcentrations of the single stranded target DNA and greater the amountof probe in the random coil conformation, the more the equilibrium K_(c)will shift toward the formation of the probe-target hybrid product,thereby increasing the fluorescent signal. However, at a given PCR cyclenumber and therefore at a given concentration of the single strandedtarget DNA, the amount of hybrid formation is proportional to theconcentration of the probe in random conformation, which is inequilibrium with the beacon in the closed conformation. Therefore, thevery existence of the closed beacon conformation reduces theconcentration of the probe in random conformation that can form thehybrid product and this reduces the strength of the fluorescent signal.Additionally, juxtaposing a pair of dyes at the probe terminals to forma dimer is likely to increase the melting temperature of the stem-loopstructure, further stabilizing the closed conformation and makingformation of the fluorescent probe-target hybrid even more unfavorable.The fluorescent signal of a beacon probe can be improved if an enzymewith 5′-exonuclease activity is used in the reaction; followingprobe-target hybrid formation, 5′-exonuclease cleaves the probe andgenerates irreversibly stable fluorescent signal. Still, the equilibriumbetween the closed and random conformations of the beacon slows down therate at which the fluorescent product is formed. Within the time frameof each PCR cycle, typically 10-30 seconds, only a fraction ofthermodynamically allowable amount of cleaved product is formed,resulting in a relatively weak signal as shown in FIGS. 6A, 6B and 6C(details in Example 8). On the other hand, homo-doubly labeled probesaccording to various embodiments of the present invention stay in anopen random conformation, and therefore can readily form fluorescentprobe-target hybrids. Cleavage of the hybridized probe by 5′-exonucleasefurther enhances the signal because it produces an irreversiblyde-quenched stable fluorescent product.

A major distinction between probes of some of the embodiments of thepresent invention and probes such as the TaqMan probe is that probes ofmany embodiments of the present invention have two or more reporter dyeswhile TaqMan probes have only a single reporter dye and a singlequencher. Although the quencher itself can also be a fluorescent dye,such as TAMRA in the FAM/TAMRA donor/quencher pair, only the emission ofthe donor dye FAM is detected and only its fluorescence signal iscorrelated to the amount of DNA produced. In TaqMan assays thefluorescence of the quencher is either ignored or not even detected.Real-time PCR detection using TaqMan probes relies upon FRET-basedfluorescence quenching of the donor fluorophore to lower the backgroundsignal in the assay. Prior to hybridization to a target DNA and/orhydrolytic cleavage of the probe by 5-exonucelase activities, thefluorescence of the donor is quenched and thus no signal or very weaksignal is detected. Following probe hybridization and/or enzymaticcleavage of the probe, the fluorescence of the donor is released andthus a positive signal corresponding to an increase to the amount of DNAproduced is detected.

Because the maximum signal that can be generated using a TaqMan isproduced by a single donor molecule the net signal gain, (the ultimateperformance) of a TaqMan probe is largely determined by the efficiencyof fluorescence quenching before and the fluorescence yield of thefluorophore after probe cleavage. Therefore, ideally in the TaqManassay, the quencher should completely quench the fluorescence of thedonor until the reporter is cleaved from the oligonucleotide. Completequenching is usually required to ensure that the real-time PCR assaystarts with a dark background. As discussed earlier, in accordance withto the well-known principles of FRET, the efficiency of FRET-basedquenching is positively related to the overlap of the emission spectrumof the donor molecule and absorption spectrum of the acceptor (quencher)molecule. For a more thorough discussion of FRET the reader is directedto references such as Förster, Ann. Phys. (1948); Stryer et al., andProc. Natl. Acad. Sci. (1967).

Furthermore considering that the fluorescence emission wavelength of adye is always longer than its absorption wavelength (as defined by itsStokes shift) the best quencher molecule for a given dye molecule willnecessarily be a different dye. This is so because the absorptionspectra of the quencher must match with the emission spectra of thedonor. In fact, the larger the Stokes shift of the donor dye is, thebetter the donor dye is because the donor can then be excited at itsabsorption maximum without having it interfere with its emission. Thisis the rationale for a second method used to increase signal output.Given the principles of FRET-based quenching, the advantage of having adonor dye with a large Stokes shift, and the need to have maximalFRET-based quenching, one would have necessarily choose a quencher withan emission wavelength substantially different the emission wavelengthof the donor dye.

In sum based on the basic principles of FRET and its wide spread use inthe construction of oligonucleotide including primers and probes thecurrent art appears to teach away from various embodiments of thepresent invention.

Accordingly, the embodiments of the invention are nonobvious in view ofthe cited art as illustrated by the results obtained with probes thatwere either homo-doubly labeled with FAM or sulfonated Cy5. FAM and Cy5are two of the most widely used reporter dyes. However, neither of thesedyes produced homo-doubly labeled probes, which exhibited significantsignal changes, and low background fluorescence (Data not shown). Inpractice, in order to increase the sensitivity of TaqMan probes, mostcommercial effort has been focused on the development of more efficientquenchers particularly quenchers based on non-fluorescent dyes. Examplesof highly efficient commercially available non-fluorescent quenchersinclude azo dye-based BHQ quenchers from BioSearch, Inc., polynitrocyanine dyes from Amersham, Inc. and the rhodamine-based YSQ dyes fromMolecular Probes.

Probes labeled with two reporter dyes according to various embodimentsdo not have to be designed so as to position the dye molecules in closephysical proximity to one another. However, aggregation is more likelyto occur among probes that are labeled with multiple dye molecules andoptional quencher molecules than among the same oligonucleotidesequences labeled with a single reporter and quencher molecule.Accordingly, at least some of the oligonucleotides, labeled with atleast two signaling dyes (and optionally with one or more quenchermolecules) according to various embodiments of the invention may exhibitlow background fluorescence and high signal output upon permanentseparation of the dyes as result of the probe cleavage, because of thelarge number of signaling molecules per oligonucleotide.

Preferably, in some embodiments, oligonucleotides are labeled with aplurality of spectrally identical or similar fluorescent dyes. A mixtureof fluorescent reporter dyes may be used for labeling a particular probeas long as the dyes have similar absorption or excitation spectra sothat they can all be efficiently excited with a single excitation light.For example, a probe of the present invention may comprise both Cy3(Glen Research, Sterling, Va.) and TAMRA (Biotium, Inc. Hayward,Calif.), both of which have similar spectra and can be efficientlyexcited at 540 nm. As another example, a particular probe may compriseboth CR110, and FAM, both of which also have similar spectra and can bewell excited by the 488 nm argon laser line. Although probes labeledwith mixed dyes are relatively more difficult to synthesize, in certaincases it can be advantageous if such mixed dyes promote fluorescencequenching prior to hybridization with a target sequence. For example, aprobe of the present invention can be labeled with a mixture of one dyewith a net negative charge and another dye of similar spectrum but witha net positive charge. A mixture of dyes with opposite charges maypromote fluorescence quenching. Methods of adding charges to dyes arewell known to anyone skilled in the art. For example, negative chargescan be added to dyes by sulfonation, while positive charges can becreated on dyes by adding secondary, tertiary, or quaternized amines todyes. For a more thorough discussion of these molecules the reader isdirected to see Mujumdar et al., 1993, Bioconjugate Chem.

More preferably, probes of some embodiments of the present invention areoligonucleotides labeled with a plurality of identical fluorescent dyemolecules. Oligonucleotide probes thereof have the advantage of beingeasily and economically manufactured because dyes can be conjugated tothe oligonucleotide in a single step. Extensive research efforts weremade in the 80's to develop efficient techniques for labeling nucleicacids. These techniques have been well documented. For a more thoroughdiscussion of this subject the reader is directed to the followingreferences: Connolly et al., Nucleic Acids Res. (1985); Dreyer et al.,Proc. Natl. Acad. Sci. (1985); Nelson et al., Nucleic Acids Res. (1989);Sproat et al, Nucleic Acids Res. (1987) and Zuckerman et al., NucleicAcids Res. (1987).

Probes of existing technologies having a reporter/quencher dye pairrequire separate labeling steps and expensive reagents. For example, thefirst label, dye or quencher, is typically attached to anoligonucleotide, either by starting the oligonucleotide synthesis with aprotected dye linked to a CPG solid support, or by incorporating the dyeduring the oligonucleotide synthesis by using a dye-labeled nucleoside(or 2′-deoxynucleoside) phosphoramidite. The second quencher or dyemolecule is attached to the oligonucleotide by using a dye- orquencher-labeled nucleoside phosphoramidite during the standard oligosynthesis. Alternatively and more typically, the second dye or quenchermolecules is attached to the oligo by first incorporating an amino groupinto the oligo during the oligo synthesis and then reacting asuccinimidyl ester dye or quencher with the amine-modified oligo. Incontrast to this multi-step labeling procedure, probes of one embodimentof the present invention comprising multiple identical dyes. Labelingwith a single dye requires only a single dye-labeling step, typically bymixing in a buffer for 1˜2 hours a reactive form of the dye with anoligonucleotide containing a desired number of a reactive groups capableof reacting with the dye. Typically, reactive groups are firstincorporated into an oligonucleotide via standard phosphoramiditechemistry using commercially available reagents.

Most preferably, probes of some embodiments of the present invention areoligonucleotides labeled with two identical fluorescent dyes whosefluorescence is quenched without formation of dye aggregates. Andpreferably, the dyes are attached to the 3′- and 5′-terminals of theoligonucleotides, respectively. In one embodiment of the invention, onedye is attached to the 3′ terminal backbone phosphate via a flexiblealiphatic linker, and another identical dye attached to the 5′ terminalbackbone phosphate via another flexible aliphatic linker. The flexiblelinkers are C2 to C30 linear or branched, saturated or unsaturatedhydrocarbon chains, optionally substituted by heteroatoms, aryls, loweralkyls, lower hydroxylalkyls and lower alkoxys. Preferably, the linkersare C4 to C12 linear or branched, saturated hydrocarbon chainsoptionally substituted by heteroatoms, lower alkyls and lowerhydroxylalkyls.

In another embodiment of invention, probes labeled with a plurality ofreporter dyes and quenchers further comprise a nucleic acid bindinggroup. Examples of nucleic acid binding groups include minor grovebinders (MGB), nucleic acid interculators, and polyamines. In cases inwhich an nucleic acid binding group is incorporating into probes ofvarious embodiments of the present invention, the oligonucleotidesequence of the probes can be me made shorter and still produce a usefulsignal. Shorter probes translate into lowering manufacturing costs.Methods of incorporating a nucleic acid binding group into anoligonucleotide probe or primer have been well documented see, forexample, U.S. Pat. Nos. 5,801,155; 6,472,153; 6,486,308; 6,492,346 andnumerous publications such as Afonina et al, N. A. R. (1997); Kumar, etal., N. A. R. (1998) and Kutyavin, et al, N. A. R. (2000). Preferably,the nucleic acid binding group is a minor grove binder (MGB). Andpreferably, probes comprising a nucleic acid binding group are labeledwith two or more identical reporter dyes. More preferably, probescomprising a nucleic acid binding group are labeled with two identicaldyes that may or may not physically touch each other.

In still another embodiment of the invention, oligonucleotide probescomprise a plurality of fluorescent reporter dyes, in which at leastsome of the reporter dyes are attached to a G nucleotide or near to a Gnucleotide located within the labeled oligonucleotide. In thisarrangement the fluorescence of the dye or dye molecules nearest to theG nucleotides are quenched before the oligo hybridizes to itscomplementary sequence.

In one embodiment of the invention, the primer is a single strandedlinear oligonucleotide comprising a plurality of spectrally identical orsimilar fluorescent reporter dyes. In the absence of a target sequence,said primer assumes a random coiled conformation and is non-fluorescentor weakly fluorescent. An oligonucleotide primer in the random coiledconformation is substantially devoid of internal secondary structure.Oligonucleotides substantially devoid of internal secondary structure donot readily from secondary structures such as of stem-loops, hairpinsand the like.

Once primers labeled with photometric molecules such as fluorescentmolecules are incorporated into the amplification product thefluorophores are further separated from each other due to the moreextended conformation of the amplification product and therefore thefluorescence signal from the photometric molecule increases. When aprimer is made in accordance with the embodiments of the presentinvention the primer includes at least two fluorophores per primer.Accordingly, primers made in accordance with the present invention canbe used to assay for complimentary oligonucleotides with greatersensitivity than assays that use primers labeled with only a singlesignaling molecule.

Upon hybridization with a target sequence and subsequent incorporationinto the amplification product, the fluorescence of some of the primersmade in accordance with embodiments of the present invention increases.Accordingly, one advantage of primers of some embodiments of the presentinvention is that multiple dye molecules present on the oligos producesignal when they hybridize to their targets. As there are at least twosignaling molecules per oligonculetide the signal they produce isgreater than the signal produced by a primer that has only a single dyemolecule attached to it. This helps to make some of the primer made inaccordance with the methods of the present invention more sensitivenucleic acid detectors than primers of existing technologies thatinclude only a single signaling molecule.

The dye molecules are typically attached to the bases of nucleotides orto a combination of the 5′ terminal backbone phosphate and the bases ofan oligonucleotide via an aliphatic linker. The dye attachment sites arespaced in a manner to achieve maximal fluorescence quenching beforehybridization and maximal fluorescence following hybridization andincorporation into the amplification product. Typically, the optimalspacing between any two adjacent pair of fluorescently labelednucleotides or between the 5′ terminal and an adjacent fluorescentlylabeled nucleotide is 10 to 50 nucleotides; Preferably, the spacing isabout 15 to 30 nucleotides; Most preferably, the spacing is about 15 to25 nucleotides.

In one embodiment is a primer labeled with two spectrally identical orsimilar fluorescent dyes. Typically, one dye is attached at or near the5′ ends via a linker and another dye attached at or near the 3′ ends viaanother linker. Preferably, one dye is attached to the 5′ terminalbackbone phosphate via a linker and, with proper spacing, another dye tothe base of a nucleotide, such as a T via another linker. Typically,linker molecules are C2 to C12 linear or branched, saturated hydrocarbonchains optionally substituted by heteroatoms, aryls, lower alkyls andlower hydroxylalkyls. The two dye molecules may be separated by about 15to about 25 bases.

In one embodiment, the primer is a linear oligonucleotide labeled withtwo identical dyes with one dye attached to the 5′ terminal backbonephosphate via a linker and another dye attached to the base of anucleotide T at or near the 3′ end via another linker. Said linkers areC4 to C12 linear or branched saturated hydrocarbon chains optionallysubstituted by heteroatoms, lower alkyls and lower hydroxylalkyls.Fluorogenic primers labeled with two identical dyes according to thepresent invention are significantly easier to manufacture than primersof existing technologies. This is especially true when a donor dye and aquencher molecule need to be attached in separate steps using expensivereagents, or wherein a long, low-yielding nucleotide needs to besynthesized to form a required hairpin structure.

Similarly, primers made in accordance with various embodiments of thepresent invention are less expensive to manufacture than primers made bymany of the currently used methods. In contrast to the multi-steplabeling procedure required to synthesize primers that include labelingmolecules with different chemistries, primers comprising two identicaldyes require only a single dye-labeling step. Typically labeledoligonucleotides of various embodiments of the present invention can bemade by mixing a reactive form of the dye with a primer containing tworeactive groups capable of reacting with the dye in a suitable bufferfor 1˜2 hours. Reactive groups are first incorporated into the oligo viastandard phosphoramidite chemistry using commercially availablereagents.

A major advantage of the fluorogenic oligonucleotides according tovarious embodiments of the present invention is the freedom to usefluorescent dyes of virtually any class and any wavelengths withouthaving to match up a report dye with a particular quencher. This may beso because some embodiments appear not to rely on classic FRET-basedfluorescence quenching to produce an assay with a useable signal.Examples of suitable classes of fluorescent dyes useful in the presentinvention include, but are not limited to, coumarins, xanthene dyes,cyanines, pyrenes, styryl dyes, BODIPY dyes, stilbenes and derivativesthereof; the widely used rhodamines, fluoresceins and rhodols belong tothe class of xanthene dyes. Preferable dyes include neutral dyemolecules and dye molecules that have a delocalized positive or negativecharge. Neutral dyes are dyes that do not bear any charge or dyes thatbear an equal number of positive charges and negative charges, neutraldyes of latter type are also referred to as zwiterionic dyes. Examplesof neutral dyes include, but are not limited to, BODIPY dyes,rhodamines, zwiterionic cyanine dyes, and derivatives thereof as shownin the following representative structures:

wherein R is a reactive group.

Examples of dyes having a delocalized positive charge include, but arenot limited to, rosamines, cyanines, and derivatives thereof as shown inthe following representative structures:

wherein R is a reactive group.

Examples of dyes having a delocalized negative charge include, but arenot limited to, fluorescein and resorufin derivatives as in thefollowing representative structures:

wherein R is a reactive group.

Alternatively, oligonucleotides of some embodiments are labeled with acombination of negatively charges dyes and positively charged dyeswherein the numbers of negatively charged dyes and positively chargeddyes are in proximately 1:1 ratio. Examples of negatively charged dyesinclude fluorescein derivatives and sulfonated dyes such as thosedescribed in U.S. Pat. Nos. 5,696,157; 6,130,101; 5,268,486; and6,133,445, and in US patent applications UA2002006479A1 andUA20020077487A1. Examples of positively charged dyes include theabovementioned rosamine dyes and cyanine dyes as well as dyes modifiedwith a tertiary or quaternary amines using standard chemistry.

In another embodiment of the present invention, suitable dyes forsynthesizing said oligonucleotides include energy transfer dyes such asthose described in U.S. Pat. Nos. 5,800,996; 6,479,303B1; and6,545,164B1 as well as International Publication WO 00/13026.Combinations of probes or primers labeled with different energy transferdyes and non-energy transfer dyes can be used for multiplex detection ina single closed tube.

Suitable dyes include for use in various embodiments include, but arenot limited to, rhodamine xanthene dyes having the following structure:

-   -   wherein R₁, R₂, R₃, and R₄ are independently H, F, Cl, C1-C18        alkyl or C2-C18 alkenyl groups; R₆ is H; R₁₀, R₁₁, R₁₂, and R₁₃        are independently H, C1-C18 alkyl groups, or C2 to C18 alkenyl        groups optionally substituted with a reactive group; said        structure further includes between 0 and 4 additional saturated        or unsaturated 5 or 6 membered rings selected from the group of        rings consisting of rings that include; R₁ in combination with        R₁₁, R₂ in combination with R₁₃, R₃ in combination with R₁₀, and        R₄; each said additional ring may be substituted with one or        more lower alkyl groups; Q is CO₂ ⁻, or SO₃ ⁻, or a reactive        group; R₇ and R₈ are independently H, F Cl, or a reactive group;        and R₆ and R₉ are independently H, F, or Cl. Suitable reactive        groups for attaching these and other dyes and quenchers to        oligonucleotides include, but are not limited to electrophiles        and nucleophiles as listed in Table 2 and other groups listed in        the definitions section. Other means for attaching photometric        molecules and quenchers include aliphatic linker groups.

Still other dyes for use in various embodiments include but are notlimited to cyanine dyes with the following structure:

wherein, R₁ and R₂ are independently selected from the groups consistingof H, F, Cl, Br, CN, carboxylic acid group, carboxamide, sulfonate,sulfonamide, lower alkyl groups, or at least one additional fusedaromatic rings, said additional fused rings include atoms independentlyselected from the group consisting of: C, N, O and S, a reactive group,and lower alkoxy groups substituted with H or a reactive group; R₃ andR₄ are independently lower alkyl groups substituted with either H or areactive group; X and Y are independently selected from the groupconsisting of: O, S, NR₅ and CR₆R₇; R₅, R₆, and R₇ wherein R₅, R₆, andR₇, R₅ are independently H, C1 to C18 alkyl groups; and “bridge” iseither a methane or polymethine group. Reactive groups suitable forattaching the molecule to an oligonucleotide include but are not limitedto the nucleophilic and electrophilic groups listed in Table 2 and othergroups listed in the definitions section. In addition to variousreactive groups, suitable dyes and quenching molecules may also beattached to oligonucleotides by for example aliphatic linkers.

Alternatively, suitable dyes are energy transfer dyes wherein one of thedye pair is a rhodamine dye or cyanine dye.

EXAMPLES Example 1 Oligonucleotide Synthesis and Labeling Materials andEquipment

All anhydrous solvents and phosphoramidite reagents includingphosphoramidites of nucleosides and protected linkers were purchasedfrom Proligo, Boulder, Colo. or Glen Research, Sterling, Va. Allunlabeled and amine-modified oligonucleotides were synthesized on anExpedite 8909 oligo synthesizer by Applied Biosciences (Foster City,Calif.).

Synthesis of Unlabeled Oligonucleotides

All unlabeled oligonucleotides (primers) were synthesized by startingwith a protected nucleoside on CPG support with a glass bead pore sizeof 500 Å. Deprotection, coupling and oxidation steps were all carriedout by following standard protocols provided by the manufacturers.Cleavage of oligonucleotides from CPG support and deprotections werecarried out by incubating the CPG beads in ammonium hydroxide at 55° C.for 16-18 hours. Once removed from the solid support, theoligonucleotides were concentrated down via a SpeedVac to remove theexcess ammonia, and then purified by passing the crude products througha Sephadex G-25 column or a C18 reverse phase cartridge. Finalpurifications, if necessary, were carried out with HPLC (SeePurification below).

Synthesis of Amine-modified Oligonucleotides

Amine-modified oligonucleotides were synthesized by using aCPG-supported amino modifier and appropriate phosphoramidite reagentscontaining a protected amine during the normal automated oligosynthesis.

A variety of commercially available CPG-supported amino-modifiers withdifferent spacer arms can be used. These products allow one to introducean amino group at the 3′ end. The CPG-supported amino modifier,3′-amino-modifier C7 CPG, was used in some of the examples listed inTable 1 and it has the following structure:

(with L₁ spacer)wherein, Fmoc and DMT are protection groups for the amine and hydroxygroups, respectively, and “succinyl-lcaa” is a spacer between the solidsupport and the modifier. The base-labile Fmoc group was removed duringammonium treatment to remove oligos from CPG support. The reagentintroduces a 7-carbon branched spacer between the amine group and the3′-end phosphate. For reference purpose, we refer to this spacer as L₁.One skilled in the art can appreciate that there are many other forms ofmodifier reagents on a solid support that can be used to introduce anamine with a different spacer, or to introduce a different reactivegroup other than an amine.

Amine-containing phosphoramidite reagents include phosphoramidites ofprotected amino-deoxynucleosides and protected amino-modifiers. The mostwidely used and also least expensive phosphoramidites of protectedamino-deoxynucleosides is phosphoramidite oftrifluoroacetylamino-2′-deoxythymidine, or amino-modifier C6 dT, whichwas used for making T-modified oligonucleotides in some of the examplesgiven Table 1. Shown below is the structure of amino-modifier C6 dT:

(with L₂ spacer)

This reagent introduces a 10-atom linear aliphatic spacer between dT andthe amine group, or between dT and a dye. For reference purpose, werefer to the 10-atom spacer as L₂.

Alternatively, an amine can be introduced to the 5′-end by using aphosphoramidite of a protected amine at the last step of the automatedsynthesis. Two amino modifier reagents, 5′-amino-modifier C6-TFA and5′-amino-modifer C12, were used for synthesizing 5′-end labeledoligonucleotides shown in this disclosure. The structures are shownbelow:

For reference purpose, the linear 6-carbon spacer of 5′-amino-modifierC6-TFA is referred to as L₃, and similarly the linear 12-carbon spacerof 5′-amino-modifer C12 is referred to as L₄.

There are many other forms of modifier reagents that can be used tointroduce an amine with a different spacer, or to introduce a differentreactive group other than an amine.

Synthesis of Dye-Labeled Oligonucleotides

Labeling reactions were conducted by adding a solution of a succinimidylester dye (Biotium, Inc., Hayward, Calif.) in DMF at −40 mg/mL to anamino oligo dissolved in 0.1 M NaHCO₃ (pH 8.5) at −1 mg/ml and vortexingthe solution at room temperature for ˜2 h. The molar ratio of dye NHSester to each amino group in the oligo was about 20-40 to 1. Unreacteddye was effectively removed by a Sephadex G-25 spin column. The crudeproducts thus obtained were subject to further purification by HPLC (Seebelow).

Purification of Labeled Oligonucleotides

Labeled oligonucleotides were purified by reverse phase HPLC on aHitachi D7000 HPLC System.

Typical HPLC condition:

Column: C18 YMC ODS-A 5 um 12 nm 150×4.6 mm, or C18 Microsorb 5 um 30 nm200×4.6.

Column temperature: 45° C.Gradient: 10%B to 50% B in 20 min-30 min @ 1 ml/min. A: 100 mM TEAAPH7.0; B: 100% CH₃CN.

Determination of Degree of Labeling

The absorbance from 230 nm to 700 nm of purified dye labeledoligonucleotides was measured on a spectrophotometer, whereby A_(max)for the dye (A_(max)) and A₂₆₀ were determined. The concentration of thedye was determined by measuring A_(max) values, while theoligonucleotide concentration was calculated based on A₂₆₀ afterfactoring in the absorbance of the dye at 260 nm. The ratio of dye tooligo concentrations defines the degree of labeling (DOL). In ourexperiments, DOL for single label is close to one (e.g. FIGS. 4B & 4C)and that for doubly labeling is close to two (e.g. FIG. 4A). Based onthis calculation we conclude that the doubly or singly labeled probes orprimers detailed in current invention are generally over 90 to 95% pure.

Example 2 Monitoring of MCG Gene Amplification Using Doubly LabeledProbes

The first set of experiment in this example demonstrates the use of adoubly 6-ROX-labeled probe in RT-PCR detection of a MCG gene. Theamplifications were performed in 20 μl reaction solution containing 10mM Tris (pH 8.0), 50 mM KCl, 3.5 mM MgCl₂, 2 mM each of dNTP, and 1 unitof AmpliTaq Gold (ABI, Foster City, Calif.). A MCG gene fragment (SEQ ID25) in pTOPO plasmid was amplified with 0.5 μM forward primer5′-TCAAGAGGTGCCACGTCTCC-3′ (SEQ ID No. 4), 0.5 μM reverse primer6-CTGATCTGTCTCAGGACTCTGACACTGT-3′ (SEQ ID No. 5). A doubly 6-ROX-labeledMCG probe, 5′-(6-ROX-L₃-CAGCACAACT ACGCAGCGCC TCC(-L₁-6-ROX)-3′ (SEQ IDNo. 6, see Table 1) was used for following the reaction. The thermalregimen was set at 95° C. for 7 minutes followed by 50 cycles of15-second duration at 95° C. and 20 second duration at 60° C.Fluorescence was measured at the 60° C. step. A series of 10-folddilutions of the template was made to create titration curves of theamplification plot. FIG. 2A shows amplification plots of aforementionedreactions starting with 10⁷ copies of template (Trace 1) down to 10¹copies of template (Trace 7). Two NTC (no template control, Traces 8 and9) are also shown in the figure. The insert shows that the Ct value isreversibly correlated with the logarithm of starting copy number (Trace10).

In the second set of experiments the probe (SEQ ID No 36) was doublylabeled with non-sulfonated cyanine dyes and the experiment was carriedout at two template concentrations, 100,000 copies (Trace 151) and 0copy (Trace 152). All other reagents and conditions were the same as inthe first set of experiments. This set demonstrates the use of a doublycyanine-labeled probe in RT-PCR detection of the MCG gene.

Example 3 Monitoring of MCG and GAPDH Gene Amplification Using a ProbeDoubly Labeled with 6-CR110 from Complex Templates

Amplifications of MCG gene fragment from human genomic DNA wereperformed as in Example 2 except (1) a 6-CR110 probe, 5′-(6CR110-L₃-)CAGCACAACT ACGCAGCGCC TCC(-L₁-6-CR110)-3′ (SEQ ID No. 7, see Table 1)and (2) a series of 10-fold dilutions of human DNA were used. FIG. 2Bshows amplification plots of the reactions starting with 10⁵ copies ofhuman DNA (Trace 11) down to 10¹ copies of human DNA (Trace 15). A NTC(Traces 16) is also shown in the figure. The insert shows that the Ctvalue is reversibly correlated with the logarithm of starting copynumber (Trace 17).

Another titration (FIG. 2C) using cDNA as template was carried out asabove except that GAPDH primers, 5′-GAAGGTGAAGGTCGGAGTC-3′ (SEQ IDNo. 1) and 5′-GAAGATGGTGATGGGATTTTC-3′(SEQ ID No. 2), and a GAPDH probe,5′-(6CR110-L₃-)CAAGCTTCCCGTTCTCAGC(-L₁-6-CR110)-3′ (SEQ ID No. 21) wereused. Starting with 0.2 μl of human brain cDNA (Invitrogen, Carlsbad,Calif.), a series of 2-fold dilutions were made. All PCR reactions werecarried out in 10 μl volume. The thermal regimen was 95° C. (7-minutes),45 cycles of 95° C. (15-second) and 56° C. (20-seconds).

Example 4 Comparison of a TaqMan Probe with a Doubly Labeled Probe ofthe Present Invention

A GAPDH gene fragment was amplified from a pTOPO plasmid containingGAPDH gene fragment (SEQ ID No. 24, Table 1) with a forward primer,5′-GAAGGTGAAGGTCGGAGTC-3′ (SEQ ID No. 1, Table 1) and a reverse primer,5′-GAAGATGGTGATGGGATTTTC-3′(SEQ ID No. 2, Table 1). A TagMan probe withJOE as the reporter dye and TAMRA as the quencher (5′-(6-JOE-L₃-)CAAGCTTCCCGTTCTCAGC(-L₁-6-TAMRA)-3′; SEQ ID No. 8, See Table 1) or aprobe according to the present invention doubly labeled with 5-R6G(5′-(5-R6G-L₃-)CAAGCTTCCCGTTCTCAGC(-L₁-5-R6G)-3′; SEQ ID No. 9, SeeTable 1) was used for monitoring the reaction under reaction conditionidentical to that used in Example 2 except that the annealing/extensiontemperature was lowered to 56° C.

All amplification reactions were carried out with 1 million copies ofthe template, and concentrations of 125 nM, 250 nM and 500 nM were usedfor each probe, respectively. As shown in FIG. 3A, the signal strengthof the probe made according to the present invention is twice as strongas that of the corresponding TaqMan probe (Trace 18 vs 21, Trace 19 vs22 and Trace 20 vs. 23). R6G and JOE have comparable spectra as well assimilar fluorescence quantum yield and extinction coefficient.Therefore, the observed performance difference between the two probes isnot due to the dyes themselves but a reflection of the superior designof the probe according to the present invention.

A FAM labeled MCG TaqMan probe with FAM at the 5′ end and TAMRA at 3′end was made and compared with doubly labeled CR110 probe(5′-(6CR110-L₃-) CAGCACAACT ACGCAGCGCC TCC (-L₁-6CR110)-3′ (SEQ ID No.7) with identical sequence in PCR. FIG. 3B shows the amplification plotsof the said homo-doubly labeled CR110 probe with TaqMan probe of 1000 nM(Trace 115 vs Trace 119), 500 nM (Trace 116 vs Trace 120), 250 nM (Trace117 vs Trace 121) and 125 nM (Trace 118 vs Trace 122) respectively. Allamplifications start with one million copies of a plasmid containing MCGfragment. At saturated concentration (1000 nM), homo-doubly labeledprobe outperformed said TaqMan probe in signal strength by 60%. Doublylabeled CR110 probe uses one half of TaqMan probe in concentration toget the same signal strength.

A FAM labeled cMyc TaqMan probe was currently provided by ABI in a 20×mixture of primer and probe. The probe has undisclosed sequence andcomprised of a MGB at its 3′ end. A comparison was made between saidTaqMan probe with doubly labeled CR110 probe (5′-(6CR110-L₃-) CAGCACAACTACGCAGCGCC TCC(-L₁-6CR110)-3′ (SEQ ID No. 7). FIG. 3C shows theamplification plots of the said TaqMan probe (Trace 110) and homo-doublylabeled CR110 probe of 1000 nM (Trace 111), 500 nM (Trace 112), 250 nM(Trace 113) and 125 nM (Trace 114). All amplifications used identicalconcentrations of human brain cDNA from Invitrogen. At saturatedconcentration (1000 nM), homo-doubly labeled probe outperformed saidTaqMan probe in signal strength. Incorporation of MGB may furtherimprove the performance of the probes in current invention.

Example 5 UV/Vis Absorption Spectra of Doubly Dye-labeled and SinglyDye-labeled Oligonucleotides and S1 Nuclease Digested OligonucleotidesThereof

The purpose of this experiment is to demonstrate that there is nophysical touching between dyes in an oligonucleotide labeled with tworeporter dyes according to the present invention. In order todemonstrate that the dyes need not touch one another we synthesizedthree CR110-labeled oligonucleotides of the same sequence: 1) a GAPDHprobe doubly labeled at 3′ and 5′ (SEQ ID No. 3, Table 1); 2) a GAPDHprobe sequence singly labeled at the 5′ (SEQ ID No. 7, Table 1); and 3)a GAPDH probe sequence singly labeled at 3′ (SEQ ID No. 8, Table 1). Thetwo singly labeled oligonucleotides were made to serve as controls.

The spectra of the three labeled oligonucleotides in S1 buffer are shownin FIGS. 4A (trace 29), 4B (trace 31) and 4C (trace 33), respectively.To assess how the spectrum of the dye is also affected by themicroenvironment surrounding the dye, the labeled oligonucleotides weredigested by S1 and then spectra were taken (FIGS. 4A (Trace 28), 4B(Trace 30) and 4C (Trace 32)). Digestions were carried out by adding 20units of S1 nuclease (Promega, Madison, Wis.) to a 100 μl reactionsolution containing 250 to 500 nM probes in S1 buffer (50 mM sodiumacetate (pH 4.5), 280 mM NaCl, and 4.5 mM ZnSO₄). S1 digestion wascompleted almost instantaneously after the S1 addition, as confirmed byfollowing the UVN is absorption spectrum change. However, spectra weretaken after 1-hour incubation at 37° C. to ensure complete digestion.

As the figures show, all three labeled oligonucleotides have nearlyidentical absorption spectra in the visible wavelength range, indicatingthat the doubly labeled probe does not have dye aggregation, which istypically characterized by a significant alteration in the shape orprofile of the absorption spectrum (See Example 7). The fact that theshape of the doubly labeled probe (FIG. 4A, Trace 29) and that of thedigested probe (FIG. 4A, Trace 28) are similar is further evidence forthe lack of dye dimer formation. The slight overall wavelength shiftfrom Trace 29 to Trace 28, as opposed to a change in the shape of theabsorption peak, is due to a difference in the microenvironment thatsurrounds the dye, similar to solvent effect. As one would expect, this“solvent effect” is similar for all three labeled oligonucleotides(Trace 28 vs. Trace 29 in FIG. 4A; Trace 30 vs. Trace 31 in FIG. 4B; andTrace 32 vs. Trace 33 in FIG. 4C).

Example 6 Amplification of GAPDH Monitored with a Homo-Doubly LabeledProbe and Two Singly Labeled Control Oligonucleotides

The purpose of this experiment is to demonstrate that the superiorperformance of the probes according to the present invention is not dueto any uniqueness of the dyes employed but the novel design of theprobes. We synthesized three labeled oligonucleotides all having thesame GAPDH probe sequence: 1) GAPDH oligo doubly labeled with 5-CR110 at3′ and 5′ ends (SEQ ID No. 3, Table 1); 2) GAPDH oligo singly labeledwith 5-CR110 at 5′ end (SEQ. ID No. 10, Table 1); and 3) GAPDH oligosingly labeled with 5-CR110 at 3′ end (SEQ ID. No. 11, Table 1). Thelabeled oligonucleotides were then tested for their utilities aspotential probes for the amplification of a GAPDH gene fragment usingcondition identical to that in Example 3.

FIG. 4D shows the kinetic profiles of GAPDH gene amplification using the3 labeled oligonucleotides as potential probes. The doubly labeled probegave a typical kinetic profile (Trace 34 in FIG. 4D), whereas under thesame amplification condition the two singly labeled oligonucleotidesfailed to respond to the amplification kinetics (Traces 35 and 36 inFIG. 4D). Successful gene amplifications for all three reactions wereconfirmed by agarose gel electrophoresis using ethidium bromide as thestain. Therefore, the lack of response from the singly labeledoligonucleotides is not caused by the absence of amplification products.These results suggest that the superior performance of the probes madeaccording to various embodiments of the present invention are a resultof the novel design of the probes. The improvements are not caused bythe unique nature of the dyes or the manner at which an individual dyeis attached to the oligonucleotides, or by the interaction between thedye and oligonucleotide.

Example 7 Spectral Comparison of Homo-Doubly Labeled Probes According tothe Present Invention with Similar Molecular Beacon Probes

Here we compare the absorption spectra of probes doubly labeled with areporter dye according to the present invention with probes having aphysically touching dye pair according to prior art. The purpose is tofurther demonstrate that unlike probes of prior art, doubly labeledprobes of the present invention do not form dye aggregates.

A GAPDH stem-loop sequence having an amine group at the 3′ and 6′ endsrespectively, 5′-(Am-L₃-)CCAAGCGGCTGAGAACGGGAAGCTTGGCTTGG(-L₁-Am)-3′ wassynthesized, where the underlined nucleotides indicate the stem-formingsequences. This amine-modified sequence was then used to synthesizethree homo-doubly labeled molecular beacon probes by reacting with thesuccinimidyl esters of 6-CR110 (SEQ ID No 12, Table 1)), 6-TAMRA (SEQ IDNo 13, Table 1) and 6-ROX (SEQ ID No 14, Table 1), respectively.Similarly, three corresponding homo-doubly labeled probes according tothe present invention were made by reacting a double amine-modifiedsequence of (Am-L₃-) CAAGCTTCCC GTTCTCAGC(-L₁-Am) with the succinimidylesters of 6-CR110 ((SEQ ID No 21, Table 1), 6-TAMRA (SEQ ID No 22,Table 1) and 6-ROX (SEQ ID No 23, Table 1), respectively. The spectra ofaforementioned stem-loop probes and their counterparts according to thepresent invention were measured at −0.5 μM in 10 mM Tris buffer (pH 8.0)at 25° C. on a Shimadzu 1201 UVN is spectrophotometer. For easycomparison, spectra for each pair of a beacon probe and thecorresponding probe of this invention were shown in FIGS. 5A, 5B and 5C,respectively. All homo-doubly labeled beacon probes showed a shorterwavelength shoulder peak, which indicates dye dimer formation (Blackmanet al., 2002, Biochemistry; Packard et al., 1996, Proc. Natl. Acad.Sci.). On the other hand, probes of the present invention had spectrasimilar to those of singly labeled oligonucleotides or digested labeledoligonucleotides (See example 5)

Example 8 Signal Strength Comparison of Doubly Labeled Probes Accordingto the Present Invention with Corresponding Homo-Doubly Labeled BeaconProbes

This experiment demonstrates that homo-doubly labeled probes accordingto the present invention are several times more sensitive than thecorresponding doubly labeled beacon probes.

A GAPDH gene fragment was amplified from a pTOPO plasmid containingGAPDH gene fragment (SEQ ID 24) using primers and conditions identicalto that in Example 4. Each of the six probes from Example 7 (threehomo-doubly labeled probes of this invention and the three correspondingbeacon probes) was used to follow the amplification reaction at fourdifferent probe concentrations, 125 nM, 250 nM, 500 nM and 1 mM,respectively. Amplifications were performed in extra cycles to ensureall reactions are complete. In addition, gel-electrophoresis revealedthat equal amount of amplified PCR products were formed for all threereactions. The kinetic profiles for each pair of a homo-doubly labeledprobe of this invention and the related beacon probe are shown in FIGS.6A, 6B and 6C respectively.

The data in FIGS. 6A, 6B and 6C clearly show that probes according tothis invention are several fold more sensitive than the correspondingbeacon probes when used at the same concentration. Also shown in thefigures is that the beacon probes did not become fully saturated even at1 mM concentration while the related probes of this invention displayedsaturation at or near 250 nM. This delayed saturation is due to theequilibrium between the open and dosed beacon conformations that makesonly a fraction of the total amount of the probe available forhybridization with the target sequence at a given time (FIG. 7).

Example 9 Probes Labeled with a Mixture of FAM and CR110

This experiment demonstrates that oligonucleotides of the presentinvention can be labeled with a mixture of reporter dyes.

To synthesize a probe labeled with a single 6-FAM and a single 6-CR110,a double amine-modified GAPDH probe sequence of 5′-(Am-L₃-)CAAGCTTCCCGTTCTCAGC(-L₁-Am)-3′ was reacted with a 1:1 mixture of 6-FAM SE and6-CR110SE. The labeling reaction produced four doubly labeledproducts: 1) 5′-(6-FAM-L₃-)CAAGCTTCCC GTTCTCAGC(-L₁-6-FAM)-3′; 2)5′-(6-FAM-L₃-) CAAGCTTCCC GTTCTCAGC(-L₁-6-CR110)-3′; 3) 5′-(6-CR110-L₃-)CAAGCTTCC GTTCTCAGC(-L₁-6-FAM)-3′; 4) 5′-(6-CR110-L₃-) CAAGCTTCCCGTTCTCAGC(-L₁-6-CR110)-3′. Products were purified by C18 RP HPLC andpeaks corresponding to products 1) and 4) were identified by comparingthe HPLC retention times of individually prepared products. Peaks thathad retention times between those of product 1) and product 4) wereassigned to those of the two hetero-doubly labeled probes, and fractionswere collected and analyzed by UVN is spectroscopy. FIG. 8A shows theUV/Vis spectra of product 1) (Trace 80), products 2) and 3) (Trace 81)and product 4) (Trace 82). The spectrum of the hetero-doubly labeledprobes (products 2) and 3) falls in between those of the two homo-doublylabeled probes as one would have expected. The isolated probes were thenused as probes for the amplification of the GAPDH gene under conditionidentical to that used in Example 4. FIG. 8B shows the kinetic profilesof GAPDH gene amplification using the isolated probes.

Alternatively, a hetero-doubly labeled oligonucleotide can be made viathe traditional method for synthesizing FRET-based probes or primers byattaching the dyes in separate steps. However, the synthesis proceduredescribed in this example may serve as a rapid way of screening foroptimal dye pairs that may yield the best performance of the labeledoligonucleotides.

Example 10 Homo-doubly Labeled Primers

These experiments demonstrate the use of oligonucleotides according tothe present invention as fluorogenic primers for RT-PCR monitoring.

In a first experiment, a fluorogenic forward primer (5′-(5-CR110-L₃-)GAAGGTGAAGGTCGGAGT (-L₂-5-CR110)C-3′, SEQ No. 15, Table 1) for a GAPDHgene amplification was synthesized by reacting 5-CR110SE with adiamine-modified primer 5′-(Am-L₃-)GAAGGTGAAGGTCGGAGT(-L₂-Am)C-3′,wherein one amine is attached to the 5′ phosphate via a C6 aliphaticlinker and another amine attached to the base of No. 18 deoxynucleotidedT via a 10-atom aliphatic linker (See Example 1 for synthesis details).Amplification of the GAPDH gene was carried out using conditionsidentical to those used in Example 4 except that: 1) no probe was used;2) the forward primer was replaced with the above homo-doubly labeledfluorogenic primer; and 3) three template copy numbers were used: onemillion, one thousand and zero (control). FIG. 9A shows theamplification profiles, where Traces 87, 88, and 89 represent onemillion, one thousand copies of templates and NTC, respectively.

In a second experiment, we synthesized another doubly labeledfluorogenic primer identical to the above forward fluorogenic primerexcept that the G at the very 5′ end is omitted, (5′-(5-CR110-L₃-)AAGGTGAAGGTCGGAGT(-L₂-5-CR110)C-3′, SEQ ID No 16, table 1). Similarly,PCR reactions were carried out using the same three template copynumbers as in the first experiment. FIG. 9B shows the amplificationprofiles, where Traces 90, 91, and 92 represent one million, onethousand and zero copies of templates, respectively. The resultindicates that the successful application of the homo-doubly labeledprimers according to the present invention is not due toG-nucleotide-associated fluorescence quenching/de-quenching, the workingmechanism of the LUX primers (Nazarenko et al., 2002, Nucleic AcidResearch)

In a third experiment, we synthesized a homo-doubly labeled reverseprimer 5′-(5-CR110-L₃-)GAAGATGGTGATGGGATT(-L₃ 5-CR110)TC-3′ (SEQ No 17,Table 1) by reacting 5-CR110SE with a diamine-modified reverse primer5′-(Am-L₂-)GAAGATGGTGATGGGATT(-L₂Am)TC-3′, wherein one amine is attachedto the 5′ phosphate via a C6 aliphatic linker and another amine attachedto the base of No. 18 nucleotide dT via a 10-atom aliphatic linker.Similarly, the GAPDH gene was amplified using condition identical tothat used in the first experiment except that 1) a regular forwardprimer was used and 2) the above homo-doubly labeled reverse primer wasused. FIG. 9C shows the amplification profiles, where Traces 93, 94, and95 represent one million, one thousand and zero copies of templates,respectively. The data from the first and second experiments indicatesthat either a forward primer or a reverse primer can be fluorogenicallylabeled according to the present invention for nucleic acid detection.

To exclude the possibility that the fluorescence quenching/dequenchingof the homo-doubly labeled primers was caused by a difference in theinteraction between the dye and the oligonucleotide before and afterhybridization with the target, we synthesized two control primers, onewith a single 5′ end label (5′-(5-CR110-L₃-)GAAGGTGAAG GTCGGAGTC-3′, SEQNo 18, Table 1), and another with a single 5-CR110 attached to the baseof No. 17 dT via a 10-atom linker (5′-(AAGGTGAAGGTCGGAGT(-L₂-5-CR110)C)-3′, SEQ No 19, Table 1). As FIGS. 9D and 9E show,neither the 5′-end labeled primer (Trace 97 in FIG. 9D) nor thedT-labeled primer (Trace 99 in FIG. 9E) responded to the PCR reaction,although gel electrophoresis of the end products revealed that both PCRreaction proceeded normally.

In still another control experiment, we synthesized a singly labeledforward primer with the dye 5-CR110 attached to the No. 18 dT nucleotidevia a 10-atom flexible linker (5′-GAAGGTGAAGGTCGGAGT(-L₂-5-CR110)C-3′,SEQ No 20, Table 1). This primer did respond positively to theamplification reactions as it was incorporated into PCR product, similarto the homo-doubly labeled primers in experiments 1) and 3). A possibleexplanation for this observation is that the dG nucleotide at the very5′ end may loop over to the 3′ end to quench the fluorophore as in thecase of LUX primers (FIG. 9F). To test this hypothesis, we synthesized asingly labeled forward primer with the 5′-end dG removed(5′-AAGGTGAAGGTCGGAGT(-L₂-5-CR110)C-3′, SEQ No 19, Table 1). As shown inFIG. 9E, this primer failed to respond to the amplification reaction.This result, along with the result from the second experiment in thisexample, indicates that in the absence of nucleotide G-associatedfluorescence quenching/de-quenching fluorogenic oligonucleotides of thepresent invention require at least two reporter dyes.

Example 11 SNP Typing with a Pair of Homo-Doubly Labeled Probes

Tapp et al have shown SNP typing by using a pair of TaqMan probes, eachlabeled with FAM and TET respectively for C to T transition of theestrogen receptor gene in codon 10. This experiment demonstrates a pairof AllGlo probes, labeled with CR110 or R6G, in replacement of FAM andTET respectively work equally well for this purpose.

SNP typing reactions were carried out in 20 μl reactions containing 10mM Tris (pH 8.0), 50 mM KCl, 3.5 mM MgCl₂, 2 mM each of dNTP, 1 unit ofAmpliTaq Gold (ABI, Foster City, Calif.), 0.5 μM forward primer5′-CCACGGACCATGACCATGA-3′ (SEQ ID No. 26), 0.5 μM reverse primer5′-TCTTGAGCTGCGGACGGT-3′ (SEQ ID No. 27), 0.2 μM ERcodon10C probe,5′-(6-CR110-L₄-CCAAAGCATCCGGGATGGCC(-L₁-6-CR110)-3′ (SEQ ID No. 28), 2μM ERcodon10T probe, 5′-(5-R6G-L₃-CCAAAGCATCTGGGATGGCC (-L₁-5-R6G)-3′(SEQ ID No. 29), and model plasmid DNA to be typed. The reaction profilewas set at 95° C. for 7-minutes followed by 50 cycles of 15 second at95° C. and 20 second at 60° C. Fluorescence was measured at the 60° C.step simultaneously from both FAM and TET channels. The homozygote CCmodel genotype consists of 10⁶ copies of a plasmid pER(C), a pTOPOplasmid containing an 106 by insert flanking the codon10 of estrogenreceptor gene, where the SNP is C (SEQ ID No. 30); the homozygote TTmodel genotype consists of 10⁸ copies of a plasmid pER(T), a pTOPOplasmid containing an 106 by insert flanking the codon10 of estrogenreceptor gene, where the SNP is T (SEQ ID No. 31); the hoterozygote CTgenotype consist of 0.5×10⁵ copies of pER(C) and 0.5×10⁵ copies ofpER(C). The three genotypes exhibited three distinct amplificationprofile patterns, these patterns are a follows: Homozygote CC had a highCR110 signal (Trace 131, FIG. 10A) and very low R6G signal (Trace 132,FIG. 10A); Homozygote TT has low CR110 signal (Trace 135, FIG. 10C) andhigh R6G signal (Trace 136, FIG. 10C); Heterozygote CT has mid-levelCR110 signal (Trace 133, FIG. 10B) and mid-level R6G signal (Trace 134,FIG. 10B).

Example 12 Amplification Using an Exo⁻ DNA Polymerase

This experiment demonstrates the use of an exo⁻ DNA polymerase in realtime PCR where the fluorescent signal was monitored by hybridizationinstead of cleavage of the probes. The amplifications were performed in20 μl reaction solution containing premixed buffer and Titanium Taq (BDBiosciences, Mountain View, Calif.). A HCV gene fragment (SEQ ID 32) inpTOPO plasmid was amplified with 2 μM forward primer5′-GCACGAATCCTAAACCTCAAAA-3′ (SEQ ID No. 33), 0.2 μM reverse primer5′-GGCAACAAGTAAACTCCACCAA-3′ (SEQ ID No. 34). A doubly 6-ROX-labeled HCVprobe, 5′-(6-ROX-L₃-ATCTGACCACCGCCCGGGAAC-(-L₁-6-ROX)-3′ (SEQ ID No. 35)at final 0.5 μM was used for each of the reactions. The thermal regimenwas set at 95° C. for 2 minutes followed by 50 cycles of 15-secondduration at 95° C., 20-second duration at 60° C. and 5 second durationat 72° C. Fluorescence was measured at the 60° C. step. A series of10-fold dilutions of the template was made to create titration curves ofthe amplification plot. FIG. 12 shows amplification plots ofaforementioned reactions starting with 10⁶ copies of template (Trace140) down to 1 copy of the template (Trace 146). An NTC (no templatecontrol, Traces 148) is also shown in the figure. The inset shows thatthe Ct value is reversibly correlated with the logarithm of startingcopy number (Trace 148).

All publications, patents, and patent applications cited in thisspecification are incorporated herein by reference as if each individualpublication, patent, or patent application was specifically andindividually indicated to be incorporated by reference and set forth inits entirety herein.

Unless specifically identified to the contrary, all terms used hereinare used to include their normal and customary terminology. Further,while various embodiments of diagnostic tests and medical treatmentdevices having specific components and steps are described andillustrated herein, it is to be understood that any selected embodimentcan include one or more of the specific components and/or stepsdescribed for another embodiment where possible.

Further, any theory of operation, proof, or finding stated herein ismeant to further enhance understanding of the present invention and isnot intended to make the scope of the present invention dependent uponsuch theory, proof, or finding.

While the invention has been illustrated and described in detail in thedrawings and foregoing description, the same is to be considered asillustrative and not restrictive in character, it being understood thatonly the preferred embodiments have been shown and described and thatall changes and modifications that come within the spirit of theinvention are desired to be protected. And while the invention wasillustrated using specific examples, and theoretical arguments, proteinand DNA sequences, accounts and illustrations these examples, arguments,illustrations sequences, accounts and the accompanying discussion shouldby no means be interpreted as limiting the invention. The Abstract ofthe Disclosure is included for the convenience of the persons searchingfor the document; the Abstract is not a summary of the invention itshould not be used to interpret or to limit the claims or specification.

1. A labeled oligonucleotide, comprising: an oligonucleotide sequencethat hybridizes to a target polynucleotide sequence; and at least twophotometric labeling molecules attached to said oligonucleotide; whereinsaid at least two photometric labeling molecules have excitationwavelengths that are within 15 nm of one another.
 2. The labeledoligonucleotide according to claim 1, wherein at least two of saidphotometric molecules produce a detectable change in signal when atleast two of said at least two photometric molecules are separated fromone another.
 3. The labeled oligonucleotide according to claim 2,wherein said at least two photometric molecules are separated from oneanother when said labeled oligonucleotide is cleaved at a positionbetween said at least two photometric molecules.
 4. The labeledoligonucleotide according to claim 2, wherein said at least twophotometric molecules are separated from one another when said labeledoligonucleotide hybridizes to the target sequence.
 5. The labeledoligonucleotide according to claim 2, wherein said at least twophotometric molecules are separated from one another when said labeledoligonucleotide is incorporated into a polynucleotide.
 6. The labeledoligonucleotide according to claim 2, wherein said two photometricmolecules is separated from one another when said two molecules arecleaved from said labeled oligonucleotide.
 7. The labeledoligonucleotide according to claim 1, wherein said at least twophotometric labeling molecules are fluorescent molecules.
 8. The labeledoligonucleotide according to claim 1, wherein any pair of saidphotometric labeling molecules on said oligonucleotide is separated fromone another by about 3 to about 60 nucleotides.
 9. The labeledoligonucleotide according to claim 1, wherein any pair of saidphotometric labeling molecules on said oligonucleotide is separated fromone another by about 12 to about 35 nucleotides.
 10. The labeledoligonucleotide according to claim 1, wherein any pair of saidphotometric labeling molecules on said oligonucleotide is separated fromone another by about 15 to about 25 nucleotides.
 11. The labeledoligonucleotide according to claim 1, further including: at least onequenching molecule attached to said oligonucleotide, wherein saidquenching molecule quenches a signal produced by said at least twophotometric labeling molecules when said at least two photometriclabeling molecules are attached to said oligonucleotide.
 12. The labeledaccording to claim 1, wherein at least one of said at least twophotometric labeling molecules is a fluorescent dye, selected from thegroup consisting of a dye that has delocalized positive charge, a dyethat has a delocalized negative charge, and a dye that has an equalnumber of positive and negative charges.
 13. The labeled oligonucleotideaccording to claim 1, wherein at least one of said at least twophotometric labeling molecules is a dye selected from the groupconsisting of BODIPY, a cyanine dye with delocalized positive charge anda zwiterionic cyanine dye.
 14. The labeled oligonucleotide according toclaim 1, wherein at least one of said at least two labeling molecules isxanthene dye molecule.
 15. The labeled oligonucleotide according toclaim 1, wherein at least one of said at least two photometric labelingmolecules is a non-sulfonated cyanine dye molecule.
 16. The labeledoligonucleotides according to claim 1, wherein at least one of said atleast two photometric labeling molecules is a dye selected from thegroup consisting of CR110, CR6G, TAMRA and ROX.
 17. The labeledoligonucleotide according to claim 1, wherein said oligonucleotidefurther includes at least one minor groove binder (MGB).
 18. The labeledoligonucleotide according to claim 1, including: at least one Guanidinenucleotide in said oligonucleotide, wherein at least one of said atleast two photometric labeling molecule is positioned near enough tosaid Guanidine nucleotide such that a signal produced by said at leastone labeling molecule is quenched when said oligonucleotide is nothybridized to said target oligonucleotide.
 19. The labeledoligonucleotide according to claim 1, wherein said oligonucleotide has a5′-end and a 3′-end; wherein one of said at least two photometriclabeling molecules is attached to the said 5′-end of saidoligonucleotide; wherein another of said at least two photometriclabeling molecules is attached to the 3′-end of said oligonucleotide.20. The labeled oligonucleotide according to claim 16, wherein saidphotometric labeling molecules attached to said 5′-end and said 3′-endof said oligonucleotide molecule are attached to said oligonucleotide byflexible aliphatic linkers.
 21. The oligonucleotide according to claim1, wherein said oligonucleotide sequence is substantially devoid ofinternal secondary structure.
 22. The oligonucleotide according to claim1, wherein said oligonucleotide is suitable for use as a probe.
 23. Theoligonucleotide according to claim 1, wherein said oligonucleotide issuitable for use as a primer in a polynucleotide amplification reaction.24. The oligonucleotide according to claim 23, wherein saidpolynucleotide amplification reaction is selected from the groupconsisting of PCR, multiplex PCR; real time PCR, quantitative PCR andreal time quantitative PCR.
 25. A method of labeling an oligonucleotide,comprising: providing an oligonucleotide sequence that hybridizes to atarget polynucleotide sequence; and attaching at least two photometriclabeling molecules to said oligonucleotide, wherein said at least twophotometric labeling molecules have excitation wavelengths that arewithin 15 nm of one another.
 26. The method of labeling anoligonucleotide according to claim 25, wherein said at least twophotometric labeling molecules are fluorescent molecules.
 27. The methodof labeling an oligonucleotide according to claim 25, wherein any pairof said photometric labeling molecules on said oligonucleotide isseparated from one another by about 3 to about 60 nucleotides.
 28. Themethod of labeling an oligonucleotide according to claim 25, wherein anypair of said photometric labeling molecules on said oligonucleotide isseparated from one another by about 12 to about 35 nucleotides.
 29. Themethod of labeling an oligonucleotide according to claim 25, wherein anypair of said photometric labeling molecules on said oligonucleotide isseparated from one another by about 15 to about 25 nucleotides.
 30. Themethod of labeling an oligonucleotide according to claim 25, furtherincluding: attaching at least one quenching molecule to saidoligonucleotide, wherein said quenching molecule quenches a signalproduced by said at least two photometric labeling molecules when saidat least two photometric labeling molecules are attached to saidoligonucleotide.
 31. The method of labeling an oligonucleotide accordingto claim 25, wherein at least one of said at least two photometriclabeling molecules is a fluorescent dye, selected from the groupconsisting of a dye that has delocalized positive charge, a dye that hasa delocalized negative charge, and a dye that has an equal number ofpositive and negative charges.
 32. The method of labeling anoligonucleotide according to claim 25, wherein at least one of said atleast two photometric labeling molecules is a dye selected from thegroup consisting of BODIPY, a cyanine dye with delocalized positivecharge and a zwiterionic cyanine dye.
 33. The method of labeling anoligonucleotide according to claim 25, wherein at least one of said atleast two labeling molecules is xanthene dye molecule.
 34. The method oflabeling an oligonucleotide according to claim 25, wherein at least oneof said at least two photometric labeling molecules is a non-sulfonatedcyanine dye molecule.
 35. The method of labeling an oligonucleotideaccording to claim 25, wherein at least one of said at least twophotometric labeling molecules is a dye selected from the groupconsisting of CR110, CR6G, TAMRA and ROX.
 36. The method of labeling anoligonucleotide according to claim 25, wherein said method furtherincludes attaching at least one minor groove binder (MGB) to saidoligonucleotide.
 37. The method of labeling an oligonucleotide accordingto claim 25, including providing at least one Guanidine nucleotide insaid oligonucleotide, wherein at least one of said at least twophotometric labeling molecule is positioned near enough to saidGuanidine nucleotide such that a signal produced by said at least onelabeling molecule is quenched when said oligonucleotide is nothybridized to said target oligonucleotide.
 38. The method of labeling anoligonucleotide according to claim 25, wherein said oligonucleotide hasa 5′-end and a 3′-end further comprising: attaching one of said at leasttwo photometric labeling molecules to the said 5′-end of saidoligonucleotide; and attaching another of said at least two photometriclabeling molecules to the 3′-end of said oligonucleotide.
 39. The methodof labeling an oligonucleotide according to claim 25, wherein saidphotometric labeling molecules attached to said 5′-end and said 3′-endof said oligonucleotide molecule are attached to said oligonucleotide byflexible aliphatic linkers.
 40. The method of labeling anoligonucleotide according to claim 25, wherein said oligonucleotidesequence is substantially devoid of internal secondary structure. 41.The method of labeling an oligonucleotide according to claim 25, furthercomprising using said oligonucleotide as a primer probe.
 42. The methodof labeling an oligonucleotide according to claim 25, further comprisingusing said oligonucleotide as a primer in a polynucleotide amplificationreaction.
 43. A kit for labeling an oligonucleotide, comprising: anoligonucleotide sequence suitable for hybridizing to a targetpolynucleotide sequence; and at least two photometric labeling moleculessuitable for attachment to said oligonucleotide; wherein said at leasttwo photometric labeling molecules have excitation wavelengths that arewithin 15 nm of one another.
 44. The kit for labeling an oligonucleotideaccording to claim 43, wherein at least one of said at least twophotometric labeling molecule are selected from the group consisting ofa dye that has delocalized positive charge, a dye that has a delocalizednegative charge, and a dye that has an equal number of positive andnegative charges.
 45. The kit for labeling an oligonucleotide accordingto claim 43, wherein at least one of said at least two photometriclabeling molecule are selected from the group consisting of BODIPY, acyanine dye with delocalized positive charge and a zwiterionic cyaninedye.
 46. The kit for labeling an oligonucleotide according to claim 43,wherein at least one of said at least two photometric labeling moleculesis a xanthene dye molecule.
 47. The kit for labeling an oligonucleotideaccording to claim 43, wherein at least one of said at least twophotometric labeling molecule is a non-sulfonated cyanine dye molecule.48. The kit for labeling an oligonucleotide according to claim 43,wherein at least one of said at least two photometric labeling moleculesis a dye selected from the group consisting of CR110, CR6G, TAMRA andROX.
 49. The kit for labeling an oligonucleotide according to claim 43,wherein said kit further includes a minor groove binder (MOB).
 50. Amethod of using a labeled oligonucleotide comprising the steps of:providing a labeled oligonucleotide, wherein said labeledoligonucleotide includes an oligonucleotide sequence suitable forhybridizing to a target polynucleotide sequence; at least twophotometric labeling molecules, wherein said at least two photometriclabeling molecules have excitation wavelengths that are within 15 nm ofone another and, wherein said photometric labeling molecules areattached to said oligonucleotide; contacting said labeledoligonucleotide with a sample.
 51. The method of using a labeledoligonucleotide according to claim 50, wherein said sample is selectedfrom the group consisting of tissue extract, cell extract, bodily fluid,in vitro nucleic acid synthesis reaction, and PCR reaction mixture. 52.A method of using a labeled oligonucleotide according to claim 50,wherein said labeled oligonucleotide is used in a nucleic acidamplification reactions wherein the reaction is selected from the groupconsisting of PCR, Multiplex PCR, Real Time PCR, Quantitative PCR, andReal Time Quantitative PCR.
 53. A method of using a labeledoligonucleotide according to claim 50, wherein said oligonucleotide is aprobe for identifying the presence of a target sequence of nucleic acidpolymer in the sample.
 54. A method of using a labeled oligonucleotideaccording to claim 50, wherein said sample is applied to a nucleic acidchip.